Dicer-2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Dicer-2
Cytological map position - 54C10
Function - enzyme
Keywords - double stranded RNA interference (RNAi), RNA-induced silencing complex (RISC)
Symbol - Dcr-2
FlyBase ID: FBgn0034246
Genetic map position - 2R
Classification - DEAD/DEAH box helicase
Cellular location - cytoplasmic
|Recent literature||Wang, Z., Wu, D., Liu, Y., Xia, X., Gong, W., Qiu, Y., Yang, J., Zheng, Y., Li, J., Wang, Y. F., Xiang, Y., Hu, Y. and Zhou, X. (2015). Drosophila Dicer-2 has an RNA interference-independent function that modulates Toll immune signaling. Sci Adv 1: e1500228. PubMed ID: 26601278
Dicer-2 is the central player for small interfering RNA biogenesis in the Drosophila RNA interference (RNAi) pathway. Intriguingly, Dicer-2 has an unconventional RNAi-independent function that positively modulates Toll immune signaling, which defends against Gram-positive bacteria, fungi, and some viruses, in both cells and adult flies. The loss of Dicer-2 expression makes fruit flies more susceptible to fungal infection. Dicer-2 posttranscriptionally modulates Toll signaling because Dicer-2 is required for the proper expression of Toll protein but not for Toll protein stability or Toll mRNA transcription. Moreover, Dicer-2 directly binds to the 3' untranslated region (3'UTR) of Toll mRNA via its PAZ (Piwi/Argonaute/Zwille) domain and is required for protein translation mediated by Toll 3'UTR. The loss of Toll 3'UTR binding activity makes Dicer-2 incapable of promoting Toll signaling. These data indicate that the interaction between Dicer-2 and Toll mRNA plays a pivotal role in Toll immune signaling. In addition, Dicer-2 is also required for the Toll signaling induced by two different RNA viruses in Drosophila cells. Consequently, these findings uncover a novel RNAi-independent function of Dicer-2 in the posttranscriptional regulation of Toll protein expression and signaling, indicate an unexpected intersection of the RNAi pathway and the Toll pathway, and provide new insights into Toll immune signaling, Drosophila Dicer-2, and probably Dicer and Dicer-related proteins in other organisms.
|Harrington, A. W. and Steiniger, M. (2016). Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells. Fly (Austin): [Epub ahead of print]. PubMed ID: 26986720
Generally, transposons move via one of two mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Recently work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. The data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. This previous analysis has now been extended to TIR Tns. In contrast to retrotransposons, the data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells.
|Wood, J.G., Jones, B.C., Jiang, N., Chang, C.,
Hosier, S., Wickremesinghe, P., Garcia, M., Hartnett, D.A., Burhenn, L.,
Neretti, N. and Helfand, S.L. (2016). Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend life span in Drosophila. Proc
Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27621458
Transposable elements (TEs) are mobile genetic elements, highly enriched in heterochromatin, that constitute a large percentage of the DNA content of eukaryotic genomes. Aging in Drosophila melanogaster is characterized by loss of repressive heterochromatin structure and loss of silencing of reporter genes in constitutive heterochromatin regions. Using next-generation sequencing, this study found that transcripts of many genes native to heterochromatic regions and TEs increased with age in fly heads and fat bodies. A dietary restriction regimen, known to extend life span, represses the age-related increased expression of genes located in heterochromatin, as well as TEs. A corresponding age-associated increase in TE transposition in fly fat body cells was also observed that was delayed by dietary restriction. Furthermore, manipulating genes known to affect heterochromatin structure, including overexpression of Sir2, Su(var)3-9, and Dicer-2, as well as decreased expression of Adar, mitigate age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also leads to an increase in life span. Mutation of Dicer-2 leads to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC results in decreased TE transposition as well as increased life span in TE-sensitized Dicer-2 mutants. Together, these data support the retrotransposon theory of aging, which hypothesizes that epigenetically silenced TEs become deleteriously activated as cellular defense and surveillance mechanisms break down with age. Furthermore, interventions that maintain repressive heterochromatin and preserve TE silencing may prove key to preventing damage caused by TE activation and extending healthy life span.
|Kandasamy, S. K. and Fukunaga, R. (2016). Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production. Proc Natl Acad Sci U S A 113(49): 14031-14036. PubMed ID: 27872309
The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) . In Drosophila, Dicer-1 produces approximately 22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. This study reports that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. It is proposed that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
|Russo, J., Harrington, A. W. and Steiniger, M. (2015).Antisense transcription of retrotransposons in Drosophila: The origin of endogenous small interfering RNA precursors. Genetics [Epub ahead of print]. PubMed ID: 26534950
To repress transposons and combat genomic instability, eukaryotes have evolved several small RNA mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. This study shows that most transposon families are transcribed in both the sense and antisense direction. LTR retrotransposons are generated from intra element transcription start sites with canonical RNA polymerase II promoters. Retrotransposon antisense transcripts were shown to be less polyadenylated than sense transcripts, which may promote nuclear retention of antisense transcripts and the double-stranded RNAs they form. Dicer-2 RNAi-depletion causes a decrease in the number of esiRNAs mapping to retrotransposons. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.
|Harrington, A.W., McKain, M.R., Michalski, D.,
Bauer, K.M., Daugherty, J.M. and Steiniger, M. (2017). Drosophila
melanogaster retrotransposon and
inverted repeat-derived endogenous siRNAs are differentially processed
in distinct cellular locations. BMC Genomics 18: 304. PubMed ID:
Endogenous small interfering (esi)RNAs repress mRNA levels and retrotransposon mobility in Drosophila somatic cells by poorly understood mechanisms. 21 nucleotide esiRNAs are primarily generated from retrotransposons and two inverted repeat (hairpin) loci in Drosophila culture cells in a Dicer2 dependent manner. Additionally, proteins involved in 3' end processing, such as Symplekin, CPSF73 and CPSR100, have been recently implicated in the esiRNA pathway.
This study presents evidence of overlap between two essential RNA metabolic pathways: esiRNA biogenesis and mRNA 3' end processing. A nucleus-specific interaction between the essential esiRNA cleavage enzyme Dicer2 (Dcr2) and Symplekin, a component of the core cleavage complex (CCC) required for 3' end processing of all eukaryotic mRNAs, was identified. This interaction is mediated by the N-terminal 271 amino acids of Symplekin; CCC factors CPSF73 and CPSF100 do not contact Dcr2. While Dcr2 binds the CCC, Dcr2 knockdown does not affect mRNA 3' end formation. RNAi-depletion of CCC components Symplekin and CPSF73 causes perturbations in esiRNA abundance that correlate with fluctuations in retrotransposon and hairpin esiRNA precursor levels. esiRNAs generated from retrotransposons and hairpins have distinct physical characteristics including a higher predominance of 22 nucleotide hairpin-derived esiRNAs and differences in 3' and 5' base preference. Additionally, retrotransposon precursors and derived esiRNAs are highly enriched in the nucleus while hairpins and hairpin derived esiRNAs are predominantly cytoplasmic similar to canonical mRNAs. RNAi-depletion of either CPSF73 or Symplekin results in nuclear retention of both hairpin and retrotransposon precursors suggesting that polyadenylation indirectly affects cellular localization of Dcr2 substrates. Together, these observations support a novel mechanism in which differences in localization of esiRNA precursors impacts esiRNA biogenesis. Hairpin-derived esiRNAs are generated in the cytoplasm independent of Dcr2-Symplekin interactions, while retrotransposons are processed in the nucleus.
|Kandasamy, S. K., Zhu, L. and Fukunaga, R. (2017). The C-terminal dsRNA-binding domain of Drosophila Dicer-2 is crucial for efficient and high-fidelity production of siRNA and loading of siRNA to Argonaute2. RNA [Epub ahead of print]. PubMed ID: 28416567
Drosophila Dicer-2 efficiently and precisely produces 21-nt siRNAs from long double-stranded RNA (dsRNA) substrates and loads these siRNAs onto the effector protein Argonaute2 for RNA silencing. The functional roles of each domain of the multi-domain Dicer-2 enzyme in the production and loading of siRNAs are not fully understood. This suty characterized Dicer-2 mutants lacking either the N-terminal helicase domain or C-terminal dsRNA-binding domain (CdsRBD) (ΔHelicase and ΔCdsRBD, respectively) in vivo and in vitro. ΔCdsRBD Dicer-2 was found to produce siRNAs with lowered efficiency and length-fidelity, producing a smaller ratio of 21 nt siRNAs and higher ratios of 20 nt and 22 nt siRNAs in vivo and in vitro. It was also found that ΔCdsRBD Dicer-2 cannot load siRNA duplexes to Argonaute2 in vitro. Consistent with these findings, DeltaCdsRBD Dicer-2 causes partial loss of RNA silencing activity in vivo. Thus, Dicer-2 CdsRBD is crucial for the efficiency and length fidelity in siRNA production and for siRNA loading. Together with previous findings, it is proposed that CdsRBD binds the proximal body region of a long dsRNA substrate whose 5'-monophosphate end is anchored by the phosphate-binding pocket in the PAZ domain. CdsRBD aligns the RNA to the RNA cleavage active site in the RNaseIII domain for efficient and high-fidelity siRNA production. This study reveals multi functions of Dicer-2 CdsRBD and sheds light on the molecular mechanism by which Dicer-2 produces 21 nt siRNAs with a high efficiency and fidelity for efficient RNA silencing.
|Harrington, A. W., McKain, M. R., Michalski, D., Bauer, K. M., Daugherty, J. M. and Steiniger, M. (2017). Drosophila melanogaster retrotransposon and inverted repeat-derived endogenous siRNAs are differentially processed in distinct cellular locations. BMC Genomics 18(1): 304. PubMed ID: 28415970
Endogenous small interfering (esi)RNAs repress mRNA levels and retrotransposon mobility in Drosophila somatic cells. esiRNAs are primarily generated from retrotransposons and two inverted repeat (hairpin) loci in a Dicer2 dependent manner. Additionally, proteins involved in 3' end processing, such as Symplekin, CPSF73 and CPSF100, have been implicated in the esiRNA pathway. This study presents evidence of overlap between two essential RNA metabolic pathways: esiRNA biogenesis and mRNA 3' end processing. A nucleus-specific interaction was identified between the essential esiRNA cleavage enzyme Dicer2 (Dcr2) and Symplekin, a component of the core cleavage complex (CCC) required for 3' end processing. This interaction is mediated by the N-terminal 271 amino acids of Symplekin; CCC factors CPSF73 and CPSF100 do not contact Dcr2. While Dcr2 binds the CCC, Dcr2 knockdown does not affect mRNA 3' end formation. RNAi-depletion of CCC components Symplekin and CPSF73 causes perturbations in esiRNA abundance that correlate with fluctuations in retrotransposon and hairpin esiRNA precursor levels. RNAi-depletion of either CPSF73 or Symplekin results in nuclear retention of both hairpin and retrotransposon precursors suggesting that polyadenylation indirectly affects cellular localization of Dcr2 substrates. Together, these observations support a novel mechanism in which differences in localization of esiRNA precursors impacts esiRNA biogenesis. Hairpin-derived esiRNAs are generated in the cytoplasm independent of Dcr2-Symplekin interactions, while retrotransposons are processed in the nucleus.
|Tants, J. N., Fesser, S., Kern, T., Stehle, R., Geerlof, A., Wunderlich, C., Juen, M., Hartlmuller, C., Bottcher, R., Kunzelmann, S., Lange, O., Kreutz, C., Forstemann, K. and Sattler, M. (2017). Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference. Nucleic Acids Res 45(21):12536-12550. PubMed ID: 29040648
RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing it was demonstrated that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.
|Coll, O., Guitart, T., Villalba, A., Papin, C., Simonelig, M. and Gebauer, F. (2018). Dicer-2 promotes mRNA activation through cytoplasmic polyadenylation. RNA 24(4):529-539. PubMed ID: 29317541
Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs, the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. This study identified Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. r2d2 mRNA, encoding a Dicer-2 partner in RNAi, was identified as a Dicer-2 polyadenylation target. These results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.
|Poirier, E. Z., Goic, B., Tome-Poderti, L., Frangeul, L., Boussier, J., Gausson, V., Blanc, H., Vallet, T., Loyd, H., Levi, L. I., Lanciano, S., Baron, C., Merkling, S. H., Lambrechts, L., Mirouze, M., Carpenter, S., Vignuzzi, M. and Saleh, M. C. (2018). Dicer-2-dependent generation of viral DNA from defective genomes of RNA viruses modulates antiviral immunity in insects. Cell Host Microbe 23(3): 353-365.e358. PubMed ID: 29503180
The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. This study found that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. It is suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.
|Sinha, N. K., Iwasa, J., Shen, P. S. and Bass, B. L. (2018). Dicer uses distinct modules for recognizing dsRNA termini. Science 359(6373): 329-334. PubMed ID: 29269422
Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, this study used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, this study found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. The study further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. These studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.
RNA silencing phenomena, either the regulation of mRNA translation or regulation of mRNA degradation, intersect at the ribonuclease Dicer. In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. siRNAs and miRNAs then direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). Mutations have been characterized in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of micro RNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 (but not Dicer-2) is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila (Lee, 2004).
Small RNAs influence a wide variety of biological processes by silencing the expression of genes within organisms. These RNAs, with a size of about 22 nucleotides, influence development, genome organization, viral and transposon defense, and disease (Hannon, 2002). There are two classes of small RNAs, and they exert their powers of silencing differently. One class is processed from longer double-stranded (dsRNA) precursor molecules with perfect complementarity. The dsRNAs are cleaved into small interfering RNAs (siRNAs) that are 21-23 nucleotide duplexes. They act as guides for a siRNA-induced silencing complex (siRISC) to target complementary mRNAs. If such an mRNA molecule is found, the base pairing interactions between siRNA and mRNA lead to cleavage of the mRNA molecule and its degradation. A second class of small RNAs, the microRNAs (miRNAs), is processed from stem-loop RNA precursors (pre-miRNAs) that are encoded within plant and animal genomes. The known functions of a few of these miRNAs indicate that they play widespread roles in growth and development (Abrahante, 2003; Brennecke, 2003; Lee, 1993; Lin, 2003; Llave, 2002; Palatnik, 2003; Reinhart, 2000). Animal miRNAs silence gene expression primarily by blocking the translation of mRNA transcripts into protein. They act as guides for a multiprotein complex, miRISC, which identifies mRNAs with imperfect complementarity in the 3' untranslated region of the message (Lee, 2004 and reference therein).
The extent of base pairing between small RNA and mRNA determines the outcome of silencing. An miRNA will direct mRNA cleavage if the target transcript is perfectly complementary in sequence (Hutvagner, 2002). Conversely, an siRNA will block protein synthesis if the target transcript has partial complementarity (Doench, 2003; Zeng, 2003). These observations imply that the extent of base pairing between small RNA and mRNA determines the outcome of silencing. It is unclear whether a single silencing complex is competent to both cleave mRNA and block translation, or whether an miRNA (or siRNA) associates with two biochemically distinct RISC complexes -- one able to cleave mRNA and another able to block translation (Lee, 2004 and reference therein).
Although dsRNAs and pre-miRNAs are structurally distinct, they are both processed into siRNAs and miRNAs, respectively, by the Dicer class of RNase III enzymes (Bernstein, 2001; Grishok, 2001; Ketting, 2001; Lee, 2002). Dicer makes staggered cuts in dsRNA to form siRNA duplexes with 3' overhangs, each strand bearing 5' phosphate and 3' hydroxyl termini (Myers, 2003; Provost, 2002). Dicer exhibits little sequence specificity for cleavage, though it favors processing from the end of a dsRNA substrate (Elbashir, 2001). The siRNA product then assembles into a siRISC that retains either the sense or antisense strand of the duplex (Hammond, 2000; Nykanen, 2001). Dicer also processes pre-miRNA (Hutvagner, 2001; Ketting, 2001; Lee, 2002). However, in the course of assembly into miRISC, one strand is preferentially retained from the siRNA-like duplex. Dicer mutants are defective for both transcript destruction and translational repression, suggesting that Dicer is required in both the siRNA and miRNA pathways (Grishok, 2001; Ketting, 2001; Knight, 2001). This dual role has made its genetic analysis more complicated (Lee, 2004 and reference therein).
In addition to dsRNA processing, Dicer appears to play some other, as yet ill-defined role in the siRNA pathway. Dicer functions downstream of siRNA production, as depletion of Dicer in mammalian cells reduces the effectiveness of added siRNAs (Doi, 2003). Dicer physically associates with protein components of RISC, and it binds siRNAs tightly in vitro (Doi, 2003; Hammond, 2001; Liu, 2003; Tabara, 2002; Tang, 2003). In Drosophila, this latter interaction is enhanced by an auxiliary dsRNA binding protein, R2D2 (Liu, 2003). Dicer-1 and Dicer-2 are shown to facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila (Lee, 2004).
Thus Dcr-1 and Dcr-2 generate different classes of small RNAs in Drosophila. Dcr-1 processes pre-miRNAs while Dcr-2 processes dsRNAs. This specificity may reflect the distinct structural properties of the two types of substrates. miRNA precursors are imperfectly paired stem loops, whereas siRNA precursors are typically long dsRNA helices with at least one blunt end. Dcr-1 might preferentially bind and attack imperfectly paired helices characteristic of miRNAs. Dcr-2 might prefer dsRNA with perfect complementarity. Another difference between siRNA and miRNA precursors is their abundance. Typically, dsRNA substrates for siRNA processing are highly abundant in cells, resulting from viral infection or promiscuous transcription. Precursors of miRNAs are expressed from endogenous genes and are not highly abundant. Dcr-1 and Dcr-2 could utilize one substrate over another according to differences in their kinetic and thermodynamic properties for each type of precursor. Finally, Dcr-1 and Dcr-2 might commit to different substrates because they contain different biochemical activities. For example, only Dcr-2 contains a DExH helicase domain and only Dcr-1 contains a PAZ domain. The PAZ domain might help link Dcr-1 to miRNA precursor molecules as they are shuttled from the cell's nucleus. These experiments clearly indicate that Dcr-2 DExH helicase activity is required for dsRNA processing. The RNA helicase domain might be needed to move Dcr-2 along the dsRNA substrate or displace Dcr-2 from the dsRNA substrate upon cleavage (Lee, 2004).
In contrast to their processing specificities, both Dcr-1 and Dcr-2 are required for siRNA-directed transcript cleavage and gene silencing. In both cases the requirement is not absolute, arguing that there is some overlapping redundancy between them. Both Dcr-1 and Dcr-2 are required for assembly of siRNA into siRISC, but play distinct roles. Dcr-2 is required to form a stable siRNA-protein complex, which contains Dcr-2 and R2D2 (Liu, 2003; Pham, 2004). This complex initiates siRISC assembly. Dcr-1 has a distinct role from Dcr-2. Dcr-1 is not necessary to form a stable initiator complex but instead functions to form a stable intermediate in siRISC assembly. It is likely that Dcr-1 is directly involved since Dcr-1 protein directly associates with siRNA in initiator complexes, intermediate complexes, and assembled siRISC (Pham, 2004). It is suggested that Dcr-1 within the initiator complex facilitates the stable association of other factors and formation of an intermediate complex (Lee, 2004).
Each Dicer has distinct qualities with regards to siRISC assembly and yet they are somewhat redundant. They may function analogously to TBP binding to a TATA sequence, initiating assembly of a transcription complex. Each component alone (Dicer or siRNA) is not sufficient to start assembly, but the combination provides enough interaction energy to drive the process. Dcr-1 and Dcr-2 remain closely associated with siRNA in assembled siRISC. This could also be analogous to TBP, which remains a component of the assembled transcription complex. However, in the process of siRISC assembly, siRNA duplex is unwound and a single strand is retained. Once this strand finds a perfect mRNA complement, the transition back to a dsRNA state initiates RNA cleavage. Clearly, Dcr-2 is not directly required for siRNA duplex unwinding, since the RNA helicase activity of Dcr-2 is not necessary for siRNA-dependent mRNA cleavage. One tantalizing notion is that the nuclease activities of the Dicers are used for cleavage of the mRNA strand within the hybrid duplex. But if so, such activities do not require the RNase III activity of both Dicers, since point mutants that abolish Dcr-2 RNase III activity still promote mRNA cleavage. One possibility is that a nonconventional catalytic site within Dcr-2 cleaves the hybrid duplex, or that Dcr-1 can efficiently cleave the duplex in place of Dcr-2. Alternatively, the Dicers might hold a siRNA-mRNA duplex in place for attack by the cleavage enzyme. The position of the mRNA cleavage site is highly exact (Elbashir, 2001), corresponding to the bond precisely ten bases from the corresponding 5' end of the siRNA strand (Lee, 2004).
Genetic analysis demonstrates that Dcr-1 but not Dcr-2 is required for gene silencing by miRNAs. Loss of Dcr-2 has a profound effect on dsRNA processing but no significant effect on Drosophila development. Although this suggests that endogenous dsRNAs do not play a critical role in development, it is possible that Dcr-1 has weak dsRNA processing activity and in a Dcr-2 mutant this weak activity might process enough endogenous dsRNA to fulfill possible developmental functions. Loss of Dcr-1 derepresses miRNA target genes and causes profound changes in development and patterning. It cannot be definitely said whether Dcr-1 and mature miRNAs form effector complexes, analogous to those formed by Dcr-1 and siRNAs. However, Dicer coimmunoprecipitates with miRNAs (Lee, 2003), suggesting that a similar mechanism is at work. Because siRISC contains Dcr-2, and because Dcr-2 is dispensable for miRISC function, it argues that siRISCs and miRISCs are inherently different. This might correlate with the functional differences seen between siRISC and miRISC with regard to mRNA cleavage and translation. That said, the capacity for Dcr-1 to act in both siRNA and miRNA pathways could explain how small RNAs of either class can cleave mRNA or block its translation, depending on the degree of complementarity between the small RNA and the mRNA target (Hutvagner, 2002; Doench, 2003; Zeng, 2003). Dcr-1 could recruit a miRNA into a RISC with cleavage activity, and Dcr-1 could recruit a siRNA into a RISC that represses translation (Lee, 2004).
In Drosophila, the Dicer-2/R2D2 complex initiates RNA interference (RNAi) by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). Recent biochemical studies suggest that the Dcr-2/R2D2 complex also facilitates incorporation of siRNA into the RNA-induced silencing complex (siRISC). This study presents genetic evidence that R2D2 and Dcr-2 are both required for loading siRNA onto the siRISC complex. Consistent with this, only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with duplex siRNA. Furthermore, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting assembly of the siRISC complexes (Liu, 2006).
The current study strengthens the model that R2D2 bridges the initiation step (siRNA production) and the effector step (siRNA loading onto siRISC) of the Drosophila RNAi pathway. The assembly of the siRISC complex is abolished or diminished when Dcr-2 or R2D2 are genetically removed. The role of the Dcr-2/R2D2 complex to load siRNA onto the siRISC complex is dependent on its ability to bind siRNA. Only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, could efficiently interact with the siRNA duplex. In addition, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting siRISC assembly. Therefore, Dcr-2 and R2D2 coordinately bind siRNA and constitute the gateway for siRNA loading and RISC activation (Liu, 2006).
Depletion of Dcr-2 by RNAi also diminishes the level of R2D2 protein, whereas RNAi of R2D2 cause a modest reduction in Dcr-2 protein in S2 cells. Moreover, recombinant Dcr-2 and R2D2 proteins are produced at much higher levels when expressed jointly than individually in insect cells. Both results suggest that Dcr-2 and R2D2 proteins stabilize each other in vivo. In the current study, it was found that the level of Dcr-2 protein remains the same in r2d21 null flies, whereas both Dcr-2 and R2D2 proteins are missing in dcr-2R416X null flies. One possible explanation for the difference seen in S2 cells and flies is that the level of Dcr-2 protein may be compensated when Drosophila cells permanently lack R2D2. Therefore, it is concluded that Dcr-2 stabilizes R2D2, but the stability of Dcr-2 is largely independent of R2D2 in the fruitfly. Consistent with this, His-tagged Dcr-2, but not R2D2, recombinant proteins can be successfully produced in insect cells. This is probably because R2D2 becomes unfolded or unstable in the absence of Dcr-2 (Liu, 2006).
The results indicate that Dcr-2 and R2D2 bind siRNA coordinately. Only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with siRNA duplex in the gel-shift assay. R2D2 contains tandem dsRNA-binding domains (dsRBDs). The first, but not the second, dsRBD of R2D2 is capable of binding long dsRNA. However, both dsRBDs of R2D2 are necessary for siRNA binding by the Dcr-2/R2D2 complex. Thus, the second dsRBD of R2D2 is critical for binding to siRNA rather than long dsRNA. It is possible that, without the second dsRBD, the first dsRBD of R2D2 only possesses low affinity for the 2122-nt siRNA duplex (Liu, 2006).
It has been shown that R2D2 as well as Dcr-2 can be efficiently cross-linked to radiolabeled siRNA by ultraviolet (UV) light. Thus, both Dcr-2 and R2D2 are in close contact with siRNA strands. Dcr-2 contains an RNA helicase domain, a DUF283 domain, and a PAZ domain at the N terminus as well as tandem RNase III motifs and a dsRBD motif at the C terminus. It is unclear which of these domains physically contact siRNA. Since neither Dcr-2 nor R2D2 bind siRNA alone, it is possible that siRNA is bound at the interface between Dcr-2 and R2D2. It is also possible that association of Dcr-2 and R2D2 triggers conformational change in either or both proteins, allowing them to bind siRNA cooperatively (Liu, 2006).
It has been reported that Dcr-2 is required for siRNA-initiated RISC assembly in vivo. This study presents genetic evidence that R2D2 is also required for loading siRNA onto the siRISC complex in vivo. It is possible that the Dcr-2/R2D2 complex helps recruit the siRNA duplex to Ago2 for siRISC assembly. However, it remains unclear exactly how the Dcr-2/R2D2 complex facilitates incorporation of siRNA into the siRISC complex. While newly synthesized siRNA is double-stranded, siRNA exists as a single-stranded form in an active siRISC complex. Thus, the nascent siRNA duplex must be unwound during siRISC assembly. It is reasonable to speculate that the Dcr-2/R2D2 complex facilitates unwinding of the siRNA duplex, thereby promoting incorporation of single-stranded siRNA into the siRISC complex. Dcr-2 is a candidate for the siRNA-unwinding helicase because it carries a putative DExH helicase domain and physically contacts the siRNA end that is easier to unwind. However, two dcr-2 mutations in the helicase domain have been isolated that do not affect the siRNA-initiated RISC activity, suggesting that a functional helicase activity is not required for Dcr-2 to promote siRISC assembly. In addition, recombinant Dcr-2 or Dcr-2/R2D2 complex cannot unwind the siRNA duplex in vitro. Alternatively, the Dcr-2/R2D2 complex may recruit an unknown helicase to unwind the siRNA duplex. In Caenorhabditis elegans, the DCR-1/RDE-4 (an R2D2 homolog) complex is associated with two highly related RNA helicases, DRH-1 and DRH-2, that are necessary for RNAi. Several RNA helicases, such as Armitage and Dmp68, have also been implicated in the Drosophila RNAi pathways (Liu, 2006).
Recent studies also suggest an alternative model for separation of siRNA strands and activation of the siRISC complex. After the Dcr-2/R2D2 complex recruits duplex siRNA to Ago2, the PIWI domain of Ago2 cleaves the passenger strand and facilitates the formation of an active siRISC complex containing only the guide strand. It is likely that the orientation of siRNA binding by the Dcr-2/R2D2 complex allows Ago2 to access and cleave only one of the two siRNA strands. Therefore, the Dcr-2/R2D2 complex determines which strand of siRNA duplex becomes the guide strand or passenger strand. The two mechanistic models of siRISC assembly are not mutually exclusive. In either model, the Dcr-2/R2D2 complex plays a critical role in facilitating the strand separation of the duplex siRNA. Since not all Ago proteins possess the slicer activity, there must be more than one mechanism for siRNA loading and RISC activation (Liu, 2006).
Infection of Drosophila with Drosophila C virus triggers a transcriptional response that is dependent in part on the Jak kinase Hopscotch. Successful infection and killing of Drosophila with the insect nodavirus flock house virus is strictly dependent on expression of the viral protein B2, a potent inhibitor of processing of double-stranded RNA mediated by the essential RNA interference factor Dicer. Conversely, flies with a loss-of-function mutation in the gene encoding Dicer-2 (Dcr-2) show enhanced susceptibility to infection by flock house virus, Drosophila C virus and Sindbis virus, members of three different families of RNA viruses. These data demonstrate the importance of RNA interference for controlling virus replication in vivo and establish Dcr-2 as a host susceptibility locus for virus infections (Galiana-Arnoux, 2006).
These data demonstrate that Dicer-2 is part of a potent effector mechanism in vivo for controlling virus infection in Drosophila. Three published studies have indicated involvement of RNAi in antiviral silencing in the nematode Caenorhabditis elegans. Those studies have shown that worms with mutations in rde-1 (which encodes a member of the Argonaute family) or rde-4 (which encodes a dsRNA-binding protein facilitating the loading of siRNA onto the RISC) contain higher viral loads after infection with FHV or the rhabdovirus vesicular stomatitis virus. However, any potential benefit of RNAi for infected worms was not addressed in those studies (Galiana-Arnoux, 2006).
By using insect flock house virus (FHV) and Drosophila as a model system, it was shown that point mutations inactivating the viral protein B2 or the host protein Dicer-2 have substantial effects on viral replication and on the resistance of flies to infection. Although B2 has been shown to bind tightly to dsRNA and to prevent its cleavage by Dicer in vitro, the data have demonstrated critical involvement of B2 in countering the Dicer-2-dependent viral RNA silencing mechanism in vivo. Unfortunately, for technical reasons, an initial attempt to express both the RNA1DeltaB2 and RNA2 transgenes on a Dicer-2 mutant background, to demonstrate restoration of the accumulation of viral RNAs and a similar effect on virulence, was unsuccessful. Nevertheless, the importance of Dicer-2 in preventing accumulation of FHV RNA in vivo was formally demonstrated by showing substantial accumulation of FHV RNA1 and RNA3 in the absence of B2 in Dicer-2 mutant flies (Galiana-Arnoux, 2006).
The potent antiviral activity of the Dicer-2-dependent RNAi mechanism was further confirmed with two other insect RNA viruses, DCV and SINV, indicating that Dicer-2 has broad antiviral functions in Drosophila. In particular, the data using SINV have shown that the outcome of the infection (death versus recovery) depends on the presence of a functional Dcr-2 gene. Although focus was placed on drosophila, the findings may be relevant to other insects, including disease vectors that transmit viruses to mammals, including humans. Indeed, increased viral loads have been reported in Anopheles gambiae mosquitoes with silencing of the gene encoding the Argonaute protein AGO2, which functions together with Dicer-2 in the RNAi pathway (Galiana-Arnoux, 2006).
The results, in conjunction with the information now available on RNAi, particularly in plants lead to a proposal that in flies Dicer-2 detects and cleaves newly synthesized viral dsRNA, generating siRNA that then specifically recognizes viral RNA and 'guides' the RISC to degrade the viral RNA. That proposed mechanism is supported more strongly by data obtained using plants, in which a nucleic acid-based antiviral defense was first described. In particular, A. thaliana plants deficient in the Dicer factor DCL2 have increased susceptibility to infection by the RNA virus turnip crinkle virus, with higher viral titers and a more deleterious disease phenotype than that of wild-type plants. However, DCL2-mutant A. thaliana are as susceptible as wild-type plants to infection by two other RNA viruses, turnip mosaic virus and cucumber mosaic virus, indicating that DCL2 does not have a general function in antiviral defenses against RNA viruses (Galiana-Arnoux, 2006).
An opposite result was demonstrated for Drosophila, for which all cases tested so far have indicated that Dicer is a global antivirus defense mechanism. The difference in results obtained with plants and Drosophila probably reflects the fact that A. thaliana expresses four DCL factors, which may have partially overlapping functions. In contrast, Drosophila has two Dicer genes: Dcr-1, which controls the production of 'micro RNA' and accomplishes important developmental functions; and Dcr-2, which controls the production of siRNA and participates in the control of viral infection. In contrast, there are no indications thus far that the sole Dicer protein found in mammals, which is essential for development, participates in the control of viral infections. Instead, mammals seem to have a diverse set of cytosolic receptors (RIG-I and MDA5) and transmembrane receptors (Toll-like receptors 3, 7, 8 and 9) that recognize viral RNA or DNA and trigger antiviral responses (Galiana-Arnoux, 2006).
Despite its importance, as demonstrated here, RNAi is certainly not the sole effector mechanism controlling virus infection in flies. The modest increase in viral RNA in FHV-infected Dicer-2 mutant flies, in contrast to the considerable effect on survival, was unexpected. Further experiments will be needed to determine whether the small differences in viral RNA concentrations in whole flies reflect tissue-specific requirements for Dcr-2 and can explain the enhanced death of infected flies or if they indicate that Dicer-2 exerts an additional function beneficial to the host other than 'dicing' viral RNA (Galiana-Arnoux, 2006).
In addition to RNAi, other antiviral effector mechanisms in plants and metazoans have been characterized, including programmed cell death, which has been reported to participate in the control of viral infections and can be blocked by specific viral inhibitors such as the baculovirus caspase inhibitor p35. Furthermore infection of Drosophila with DCV triggers induction of some 150 genes by a factor of two or more. At least some of those genes encode proteins that participate in controlling the infection, an hypothesis supported by the fact that the genes are not induced after virus infection in Jak-deficient flies, mutant flies that have higher viral loads than wild-type control flies and are generally more susceptible to infection (Galiana-Arnoux, 2006).
Strictly speaking, there is at present evidence for two types of responses to virus infection in Drosophila: degradation of viral RNA by the RNAi machinery and cytokine-mediated induction of many genes (via hopscotch-encoded Jak activated by the gp130-related cytokine receptor Domeless), some of which may counter viral infection. The coexistence of those two types of response may reflect an important difference in RNAi in plants versus Drosophila: whereas RNAi is cell autonomous in Drosophila, in plants the RNAi response triggered in infected cells spreads systemically to the plant to induce protective RNAi at distant sites. That cell-to-cell transfer of the silencing signal is essential for the host to counter viral infection, as the presence of dsRNA is in most cases detected after viral replication at a stage at which the cells may not succeed in blocking or destroying the virus. It is proposed that in Drosophila, RNAi functions to limit viral replication in infected cells and is coupled to other defense mechanisms triggered by cytokine signaling in uninfected cells. A principal challenge for future work will be to elucidate how the integration of these responses allows Drosophila to resist viruses (Galiana-Arnoux, 2006).
Intrinsic immune responses autonomously inhibit viral replication and spread. One pathway that restricts viral infection in plants and insects is RNA interference (RNAi), which targets and degrades viral RNA to limit infection. To identify additional genes involved in intrinsic antiviral immunity, Drosophila cells were screened for modulators of viral infection using an RNAi library. Ars2 was identified as a key component of Drosophila antiviral immunity. Loss of Ars2 in cells, or in flies, increases susceptibility to RNA viruses. Consistent with its antiviral properties, it was found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing primary miRNA (pri-miRNAs). The identification of Ars2 as a player in these small RNA pathways provides new insight into the biogenesis of small RNAs that may be extended to other systems (Sabin, 2009).
Innate immunity is the most ancient line of defense against pathogens. In mammals, the innate immune system provides the initial response to infection and primes the adaptive immune response. In contrast, invertebrates and plants lack adaptive immunity and therefore rely solely on innate mechanisms to combat infections. Recent studies have identified RNA interference (RNAi) as an ancient, cell-intrinsic immune mechanism that controls RNA viruses in plants and insects. RNAi is one of several small RNA-dependent silencing pathways that control gene expression in a sequence-specific manner in plants and animals. Small RNA-driven silencing is initiated by an RNase III enzyme Dicer, which produces RNA duplexes of approximately 21 nucleotides through the cleavage of longer precursor molecules. Once generated, the duplex is incorporated into the multiprotein RNA-induced silencing complex (RISC) where one strand of the duplex is preferentially retained. This guide strand directs RISC to a homologous target, where an Argonaute protein (Ago) mediates posttranscriptional gene silencing (Sabin, 2009).
In Drosophila, small-interfering RNAs (siRNAs), which are derived from exogenous or endogenous sources of double-stranded RNA (dsRNA), are generated by Dicer-2 (Dcr-2) and incorporated into an Ago2-dependent RISC, leading to the degradation of complementary mRNAs or viral RNAs. In contrast, endogenous primary miRNA transcripts are processed into pre-miRNAs in the nucleus by the Microprocessor, which includes the RNase III enzyme Drosha and its binding partner Pasha. Next, the pre-miRNAs are exported and further processed by cytoplasmic Dicer-1 (Dcr-1) into mature miRNAs. Mature miRNAs are incorporated into an Ago1-dependent RISC and mediate translational inhibition of target transcripts (Sabin, 2009).
Studies using mutants in the known components of the classical siRNA pathway, including Dcr-2, r2d2, and AGO2, revealed the essential antiviral role that RNAi plays against positive-stranded RNA viruses in Drosophila. This study set out to identify additional cellular components of the Drosophila intrinsic antiviral arsenal. An unbiased screen for factors that, when lost, led to increased viral replication identified Ars2 (CG7843). Ars2 is a poorly characterized gene that is highly conserved and required for development in Arabidopsis, zebrafish, and mice. The best characterized Ars2 homolog is Arabidopsis SERRATE, which recently emerged as a component of the miRNA biogenesis pathway. In plants, Ars2 genetically interacts with the nuclear cap-binding complex (CBC) components ABH1/CBP80 and CBP20. Both thus study and the work in the accompanying manuscript now reveal a physical interaction between Ars2 and the CBC in Drosophila and mammals. It is further demonstrated that Ars2, along with the CBC, plays a role in antiviral immunity against a battery of RNA viruses and is required for both siRNA- and miRNA-mediated silencing, controlling the biogenesis of small regulatory RNAs (Sabin, 2009).
To identify novel genes that control viral replication, a small-scale screen was performed for cellular factors that allow increased viral replication in cells when depleted by RNAi. A Drosophila cell culture system was used for these studies due to the high potency of RNAi, low genomic redundancy, and lack of a complicating interferon response. To screen for such host factors, Drosophila cells with dsRNA were treated, incubated the cells for 3 days to allow for loss-of-function phenotypes, and the cells were challenged with the mammalian virus Vesicular Stomatitis Virus (VSV). VSV is an enveloped, negative-stranded RNA virus that is naturally transmitted to mammals from insects. To monitor infection, a recombinant virus was used that expresses the reporter GFP upon replication. Treatment with dsRNA against GFP is used as a positive control for silencing, as it is expressed by the virus upon replication. Luciferase dsRNA is used as a nontargeting control, as it is not expressed in this system. Using a fluorescence assay it was found that VSV readily infects Drosophila cells, and that RNAi-mediated depletion of GFP can be monitored by microscopy. Using this strategy approximately 100 genes were screened and CG7843, the Drosophila homolog of mammalian Ars2, which led to an increase in the percentage of VSV-infected cells when depleted by RNAi, was identified. To validate that the increase in infection was due to a depletion of Ars2 rather than an off-target effect, an independent dsRNA to Ars2 was generated and a similar increase in VSV infection was observed. Effective knockdown of Ars2 was verified by northern blot analysis (Sabin, 2009).
This study demonstrates that Ars2 and the cap-binding complex (CBC) are required for miRNA- and siRNA-mediated silencing as well as antiviral defense in Drosophila. Ars2 and the CBC are required at upstream steps in both Drosophila and mammalian RNA silencing pathways, consistent with recent data from plants; the Ars2 homolog SERRATE and homologs of the CBC (ABH1 and CBP20) control pri-miRNA processing in Arabidopsis. Ars2 functionally and biochemically interacts with the nuclear Microprocessor, as does SERRATE, which physically interacts with HYL1, a component of the Arabidopsis miRNA biogenesis pathway similar in function to Drosophila Pasha and mammalian DGCR8 (Sabin, 2009 and references therein).
The results of these studies, combined with the evidence from existing literature, have led to the proposal of two potential models for Ars2 function. The first is a bridging model in which Ars2 serves as a recruitment factor to guide the RNA processing machinery to the proper substrates. Under this model, Ars2 and the CBC bind pri-miRNA transcripts through recognition of their 5' cap, and Ars2 actively recruits the Microprocessor to the transcript, promoting its cleavage into a pre-miRNA. This bridging model suggests a mechanism by which Ars2 and the CBC increase the efficiency of pri-miRNA processing by acting as chaperones to stabilize and deliver the primary transcripts directly to the Microprocessor. It then follows that in the absence of Ars2 or the CBC, Drosha-directed pri-miRNA processing is impaired since the recruitment of the Microprocessor to primary transcripts is less efficient, and the unprocessed transcripts are destabilized. The finding that pri-bantam levels are reduced in Ars2 or CBC-depleted cells along with similar findings in mammalian cells (Gruber, 2009) support this model. While the bridging model provides a compelling mechanism for the Ars2 and CBC requirement in the miRNA pathway, it is less likely to be relevant for the siRNA pathway, as the substrates of the pathway (dsRNA, viral RNAs) are not necessarily 5' capped. Although it is possible that Ars2 recruits Dcr-2 to uncapped substrates through the targeting of secondary RNA structure, the data favor an RNA recognition-independent model (Sabin, 2009).
This second model proposes that Ars2 serves as a cofactor for the enzymatic activity of RNase III enzymes. Under this model, the presence of Ars2 in Drosha- or Dcr-2-containing complexes promotes robust enzymatic cleavage of RNA substrates and increases the fidelity of processing. Consistent with this model, recent work by Dong has shown that the addition of recombinant SERRATE to an in vitro pri-miRNA processing assay enhances both the activity and the accuracy of DCL1 substrate cleavage. Moreover, Gruber demonstrate in the accompanying manuscript that mammalian pri-miRNA processing is altered in the absence of Ars2 (Gruber, 2009). Functional dicing assay demonstrates that Dcr-2-mediated processing of long dsRNA is impaired in the absence of Ars2, lending support to the idea that Ars2 is an essential accessory factor for Dcr-2 activity on uncapped RNAs. Since the substrates of the cytoplasmic siRNA pathway are not necessarily capped, this further argues that the CBC may be required for the cofactor activity of Ars2 rather than for substrate recognition (Sabin, 2009).
The underlying difference between the two models is the precise step of substrate recognition and processing for which Ars2 is required; the bridging model poses that Ars2 and the CBC physically bind the RNA substrate, recruiting the proper processing activity for cleavage. Conversely, the cofactor model suggests that the binding of Ars2 and the CBC to the processing machinery allows the enzymes to execute robust and accurate substrate cleavage. Of course, these models are not mutually exclusive, and the true function of Ars2 may combine aspects of both models. Ars2 may also play distinct roles depending on its particular binding partners or intracellular localization. Ultimately, this study implicates Ars2 as a fundamental component of several modes of RNA silencing and contributes to the growing body of evidence that RNA silencing pathways are more interconnected than previously appreciated. The RNA content of a cell is influenced by the contributions of many transcriptional and posttranscriptional regulatory pathways that have evolved to respond quickly and sensitively to the needs of the cell. The identification of novel components of these pathways, such as Ars2, aids in efforts to uncover the mechanisms by which cellular processes such as proliferation or antiviral defense are exquisitely regulated (Sabin, 2009).
Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. Components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets (Grimaud, 2006).
The RNAi machinery has been implicated in a wide variety of biological processes. One of these processes is the formation of heterochromatin. In S. pombe, this involves bidirectional transcription of RNA molecules from repetitive sequences and their cleavage into short interfering RNAs (siRNAs) of 21–23 nt by an RNase III enzyme called Dicer-1. siRNAs guide the RNA-induced initiation of transcriptional gene silencing (RITS) complex to homologous sequences in the nucleus (Noma, 2004; Verdel, 2004). Clr4, the homolog of the histone methyltransferase Su(Var) 3-9, is recruited along with the RITS complex to chromatin, where it methylates lysine 9 of histone H3 (H3K9). This epigenetic mark promotes the formation of heterochromatin by recruiting the heterochromatin protein Swi6, the homolog of HP1, via its chromodomain (Grewal, 2004). Consistent with these data, a redistribution of H3K9 methylation has been observed in Drosophila chromosomes in flies mutant for components of the RNAi machinery (Pal-Bhadra, 2004; Grimaud, 2006).
The RNAi machinery is also required for cosuppression, a phenomenon whereby the introduction of multiple transgenic copies of a gene phenocopies its loss of function instead of increasing its expression. In Drosophila, cosuppression can act at either the transcriptional or posttranscriptional level and involves PcG proteins as well as the RNAi machinery (Pal-Bhadra, 1997, Pal-Bhadra, 1999 and Pal-Bhadra, 2002; Grimaud, 2006 and references therein).
The Drosophila RNAi machinery includes two Dicer proteins encoded by the dicer-1 (dcr-1) and dicer-2 (dcr-2) genes. Dcr-2 is specifically required to process double-stranded RNAs into siRNAs and mediates the assembly of siRNAs into the RNA-induced silencing complex (RISC). Dcr-1 is involved in the metabolism of siRNAs as well as the processing of pre-microRNAs into microRNAs. RNA silencing also involves several highly conserved genes coding for PAZ-domain proteins. Argonaute1 (AGO1) and Argonaute2 (AGO2) are involved in microRNA biogenesis and RNA interference (RNAi). piwi is involved in cosuppression, silencing of retrotransposons, and heterochromatin formation. aubergine (aub) was first isolated based on its role in germline development but is also responsible for maintaining the silenced state of an X-linked male fertility gene locus (Stellate) via RNAi. The Aub protein is required for RNAi and RISC assembly in ovaries. In addition, homeless/spindle-E (hls) is involved in silencing of Stellate and in heterochromatin formation. This study tested whether RNAi components are involved in the PcG pathway. The results show that the RNAi machinery affects the PcG response via a novel regulatory function in nuclear organization (Grimaud, 2006).
The role of a variety of RNAi components in a specific transgenic line called Fab-X was tested. This line contains a construct carrying a 3.6 kb fragment from the Fab-7 region, cloned upstream of a mini-white reporter and inserted into the X chromosome. In the Fab-X line, the presence of the Fab-7 sequence is sufficient to induce PcG-dependent silencing, both of the mini-white eye-color reporter gene and of the endogenous scalloped (sd) gene, which is required for wing-blade morphogenesis and is located 18.4 kb downstream of Fab-7. These two repressed phenotypes are abolished in the presence of mutations in PcG genes and are not present in heterozygous females and hemizygous males, indicating that both mini-white and sd expression are subject to PSS (Grimaud, 2006).
The eye-color and wing phenotypes were used as a basis to analyze the effect of the RNAi machinery on PcG-dependent repression. Mutations in RNAi components were introduced into the Fab-X line and placed over a balancer chromosome containing a GFP marker. As the AGO1 mutant alleles involve P element insertions containing the mini-white reporter gene, they could not be tested using the eye phenotype. A null mutation in dcr-2 (dcr-2L811fsX) decreased silencing of the mini-white reporter gene relative to the Fab-X line when in the homozygous state. Likewise, two different mutant alleles of piwi (piwi1 and piwi2) decreased mini-white silencing, with the effect being more pronounced in piwi2 mutant flies. This effect was not restricted to the Fab-X line since it was also observed when piwi2 was recombined into another Fab-7-containing line. In contrast to the effects seen for dcr-2 and piwi alleles, Fab-X females homozygous mutant for hlsE1 or that carried the heteroallelic hlsE1/hlsE616 combination silenced mini-white like wt Fab-X females (Grimaud, 2006).
The sd phenotype was then analyzed in all mutant backgrounds at 28.5°C, a temperature inducing a strong wing phenotype in Fab-X. A preselection of non-GFP female larvae was carried out in order to selectively analyze homozygous or trans-heterozygous mutant adults. This analysis revealed that mutating any of the components of the RNAi machinery, except for hls and the heterozygous dcr-1 mutation, leads to a strong decrease in the sd phenotype. These data show that the RNAi machinery can affect PcG-mediated silencing. The fact that hls mutants had no effect suggests that this process might be mechanistically distinct from the role of RNAi components in heterochromatin formation (Grimaud, 2006).
While most RNAi components are not required for binding of PcG proteins to PREs, they are required to mediate long-range contacts between multiple copies of the Fab-7 element. Moreover, Dcr-2, PIWI, and AGO1 colocalize with PH in the cell nucleus, and their effect correlates with the presence of small RNAs homologous to Fab-7 sequences. Finally, in addition to their effects on transgenic Fab-7 copies, mutations in these genes also reduce the frequency of long-distance contacts between endogenous PcG target genes. Taken together, these results reveal a novel and unexpected role for the RNAi machinery in the regulation of euchromatic genes in the nuclear space (Grimaud, 2006).
The effects caused by mutations in different RNAi components suggest the existence of distinct molecular roles for these proteins in the regulation of PcG function. First, the hls gene does not seem to play a major role in silencing at the Fab-7 PRE or maintaining long-distance Fab-7 contacts. Since Hls has been shown to play a central role in heterochromatin formation (Pal-Bhadra, 2004), there may be different subtypes of nuclear RNAi machineries for heterochromatin formation and for regulating PcG function. No effect was found when a mutation in the dcr-1 gene was analyzed at the heterozygous state, but the elucidation of the function of dcr-1 in PcG-mediated silencing awaits further analysis in a homozygous mutant background. A second class of RNAi components that participate in PcG-mediated repression contains dcr-2, AGO1, and the aub gene. Loss of any of these RNAi gene products affects PcG-dependent silencing at Fab-7, although it does not impact the binding of PcG proteins to Fab-7. Only mutations in piwi affected the binding of PcG proteins to Fab-7, at least in polytene chromosomes, but even PIWI did not affect recruitment of PcG factors at endogenous genes. In S. pombe, both RNAi components as well as DNA binding proteins are involved in recruiting heterochromatin proteins to the mating-type region (Jia, 2004). At PREs, multiple DNA binding factors and chromatin-associated proteins are known to contribute to PcG protein recruitment, such as PHO, the GAGA factor, DSP1, and the CtBP proteins. Their combinatorial action might play a key role in the robust and specific chromatin tethering of PcG proteins, while, in contrast to the situation in S. pombe, the RNAi machinery might play a relatively minor role (Grimaud, 2006).
It is interesting to note that piwi mutations affected recruitment of E(Z) and PC to Fab-7 in polytene chromosomes but had no effects on PH, another PRC1 component. In the current model for recruitment of PcG proteins to PREs, histone H3 methylation by the E(Z) protein recruits PRC1 via the chromodomain of PC. The current results indicate that multiple mechanisms might be used to anchor different PRC1 components to PREs and that the loss of PC does not necessarily lead to the disintegration of the entire PRC1 complex at PREs (Grimaud, 2006).
To date, all transcriptional gene-silencing phenomena that depend on the RNAi machinery involve the production of small-RNA molecules. RNAi components were also shown to affect telomere clustering in S. pombe, although binding of Swi6 and H3K9me to individual telomeres is not affected. The production of siRNAs is believed to be essential for the nuclear clustering of telomeres since cells carrying a catalytically dead RNA-dependent RNA polymerase (which abolishes siRNA production) are defective in telomere clustering. Consistent with a role for small RNAs in mediating gene contacts, sense and antisense transcription of Fab-7 as well as small-RNA species were found in Fab-7 transgenic lines. Moreover, a mutant allele of dcr-2 producing a truncated polypeptide lacking the RNase III domain, which is required for dsRNA processing, is defective in long-range interactions of PcG target sequences as well as in accumulation of Fab-7 small RNAs. These data suggest that small-RNA species could be involved in these gene contacts. However, no small Fab-7 RNA species was detected in the wt situation, although RNAi mutants affect the contact of the endogenous Fab-7 locus with the Antp gene. This might indicate that other RNA species produced in the endogenous Hox genes could contribute to gene clustering. However, the possibility remains that RNA-independent functions of RNAi proteins contribute to the maintenance of gene contacts, in particular in the case of endogenous PcG target genes (Grimaud, 2006).
Interestingly, none of the RNAi mutants tested are defective in the establishment of long-distance chromosomal interactions. Fab-7 contacts are correctly established during embryogenesis but decay during later stages of development. This suggests that the RNAi machinery is not required to initiate contacts but rather to maintain them via the stabilization of gene clustering at specific nuclear bodies. This clustering could be important in cosuppression, where transgene silencing can occur at the transcriptional and posttranscriptional levels, both requiring the RNAi machinery (Pal-Bhadra, 2002). It is difficult, however, to understand how a relatively modest increase in transcript levels caused by an increase in the copy number of a gene could trigger a robust silencing of all copies. This is particularly puzzling considering that the transcript levels of endogenous single-copy genes can vary, e.g., during normal physiological gene regulatory processes, without triggering gene silencing. One explanation might be that cosuppressed genes are clustered in the cell nucleus. Indeed, clustering of multiple gene copies has been reported in plant cells (Grimaud, 2006).
It is proposed that the RNAi machinery, perhaps in conjunction with PcG proteins, might stabilize this gene-clustering phenomenon. Specifically, the colocalization of multiple gene copies with components of the RNAi machinery might increase the local concentration of RNA species. Once this concentration overcomes a critical threshold, double-stranded RNAs might assemble and be cleaved in situ by the enzymatic activity of the RNAi machinery. RNA molecules might contribute to hold together loci containing PcG proteins that produce noncoding transcripts encompassing PREs. This gene clustering might involve contacts with components of the RNAi machinery as well as PcG proteins assembled in the same nuclear compartments (Grimaud, 2006).
One important question is, what is the role of the RNAi machinery in the regulation of endogenous PcG target genes? The data indicate that RNAi components affect only a subset of these genes since the colocalization of PcG bodies with RNAi bodies is limited. Hox loci are characterized by extensive noncoding RNA transcription, and, recently, other PcG target genes have been shown to be associated to intergenic transcription. RNAi components might be targeted to this subset of PcG target genes, while other PcG target genes that are characterized by the absence of noncoding transcripts might be independent on RNAi factors (Grimaud, 2006).
The fact that no homeotic phenotypes are visible in RNAi mutant backgrounds suggests that the function of RNAi components can be rescued by other chromatin factors. Indeed, the decrease in the level of nuclear interaction between the homeotic complexes was incomplete in RNAi mutant backgrounds. The data suggest that, while the RNAi machinery does not act in the establishment of PcG-dependent gene silencing, RNAi factors might help stabilize silencing during development by clustering PcG target genes at RNAi nuclear bodies. Thus, in addition to its role in defending the genome against viruses, transposons, and gene duplications, the RNAi machinery might participate in fine tuning the expression of PcG target genes through the regulation of nuclear organization. Finally, it must be noted that the developmental expression profile of the components of the RNAi machinery is highly specific. The function of specific RNAi components is therefore likely to be highly variable in different cell types and as a function of time. It will be of great interest to explore this issue in the developmental context of the whole organism, in Drosophila as well as in other species (Grimaud, 2006).
RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs were studied from viral RNA. To better characterize the substrates of Dicer-2, the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. It was found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, evidence is provided that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi (Sabin, 3013).
The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2 cells and consists of two stoichiometric subunits: Dicer-2 (Dcr-2) and a previously unknown protein that has been termed R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of Dcr-2. Rather, the Dcr-2/R2D2 complex, but not Dcr-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC (Liu, 2003).
RNA interference (RNAi) is a form of posttranscriptional gene silencing whereby double-stranded RNA (dsRNA) molecules trigger the sequence-specific degradation of cognate mRNA. The biological importance of RNAi is underscored by its wide conservation throughout metazoans and the existence of closely related systems in plants (called cosuppression) and fungi (called quelling). Emerging evidence indicates that the RNAi and related pathways function in many fundamental biological processes, including antiviral defense, development, and maintenance of genomic stability (Liu, 2003 and references therein).
The Drosophila RNAi pathway consists of initiation and effector steps. (1) Long dsRNA molecules are cleaved into 21- to 23-nucleotide (nt) small interfering RNA (siRNA) duplexes. (2) The siRNA is incorporated into a nuclease complex named RNA-initiated silencing complex (RISC) and functions as a guide RNA to direct RISC-mediated sequence-specific mRNA degradation. The endonucleases that process dsRNA have been identified as Dicers, a family of large noncanonical ribonuclease (RNase) III enzymes. Although only one Dicer enzyme is found in Caenorhabditis elegans and humans, two (Dcr-1 and Dcr-2) have been identified in Drosophila. It remains unclear how siRNA is transferred from Dicer to RISC (Liu, 2003 and references therein).
This problem was studied by purifying the siRNA-generating activity from the cytoplasmic (S100) extract of S2 cells through a six-step chromatographic procedure. A single major peak of activity was observed at all steps and was followed throughout purification. Two proteins, 190 kD and 36 kD, showed perfect correlation with the enzymatic activity after the final gel filtration step. They were identified by mass spectrometry as Dcr-2 and a previously unknown protein (Flybase CG7138), respectively. This protein was named R2D2 because it contains two dsRNA-binding domains (R2) and is associated with Dcr-2. R2D2 bears 20.9% identity and 33.4% similarity to the C. elegans RNAi protein RDE-4 (Tabara, 2002; Grishok 2000), which also contains tandem dsRNA-binding domains and interacts with Dicer (Liu, 2003).
To confirm the purification results, immunodepletion experiments were performed with antiserum directed against the carboxyl-terminal 150 amino acids of R2D2. This R2D2 antibody depleted both R2D2 and Dcr-2 proteins and removed the majority of siRNA-generating activity from S100, whereas the level of Dcr-1 remained unchanged in the supernatant. Furthermore, when S100 was fractionated on a QSepharose column, Dcr-2 and R2D2, but not Dcr-1, correlated perfectly with the siRNA-generating activity (Liu, 2003).
To determine the contribution of Dcr-1 and Dcr-2 to siRNA production in Drosophila cells, either protein from S2 cells was depleted by dsRNA soaking. Interestingly, Dcr-2 dsRNA also caused a substantial reduction in the level of R2D2 protein. This was not simply due to cross-targeting because the level of R2D2 mRNA was unaffected. Likewise, R2D2 dsRNA also reduced, although not as dramatically, the level of Dcr-2 protein. These results indicate that Dcr-2 and R2D2 form a stable complex and that either protein alone is unstable. Although depletion of Dcr-1 made no difference, knocking down Dcr-2 reduced the siRNA-generating activity by fivefold in lysates and interfered with RNAi in vivo. Together, these results demonstrate that the Dcr-2/R2D2 complex is the principal siRNA-generating enzyme responsible for initiation of RNAi in Drosophila S2 cells (Liu, 2003).
To determine whether R2D2 is required for RNAi in vivo, r2d2 deletion mutant flies were generated by P element mobilization, and they were crossed with transgenic flies expressing green fluorescent protein (GFP) under the ubiquitin promoter to derive homozygous r2d2: Ub-GFP mutant flies. Then 0- to 2-hour wild-type or r2d2 mutant embryos were collected for microinjection of GFP dsRNA. Whereas introduction of GFP dsRNA effectively silences GFP expression in wild-type embryos, r2d2 mutant embryos were completely defective for the dsRNA-initiated RNAi response (Liu, 2003).
The siRNA-generating activity was reconstituted in vitro with the use of purified His6-tagged Dcr-2 and R2D2 recombinant proteins expressed in insect cells. A mutant form of R2D2 was also created by point mutations in the dsRNA-binding domains that abolish its ability to bind dsRNA. Dcr-2 protein alone efficiently cleaves dsRNA into siRNA in an adenosine triphosphate (ATP)- and dose-dependent manner. The Dcr-2/R2D2 complex is also ATP-dependent and shows activity equivalent to Dcr-2 alone. Kinetic studies were performed with Dcr-2 and Dcr-2/R2D2 recombinant proteins and no statistically significant difference was found in their Km (a measure of substrate affinity) or Kcat (a measure of catalytic activity). Furthermore, the mutant Dcr-2/R2D2M complex was as active in siRNA production as the wild-type complex. These results indicate that association with R2D2 does not affect the ability of Dcr-2 to recruit or cleave dsRNA. This finding is inconsistent with the proposed function of RDE-4 to recruit dsRNA to Dcr-1 (the single Dicer in worms) for processing. However, R2D2 may stabilize Dcr-2 and thereby positively regulate siRNA production in Drosophila cells. Consistently, Dcr-2 and R2D2 were expressed at much higher levels in insect cells when expressed jointly than separately (Liu, 2003).
To follow the fate of siRNA, a gel shift assay was developed to identify proteins that interact with siRNA. When radiolabeled synthetic siRNA was incubated with S2 lysate, a distinct mobility shift was observed on a native polyacrylamide gel. The formation of this siRNA-protein complex did not require ATP hydrolysis because it could be carried out efficiently at 4°C. Furthermore, when S100 was fractionated by a QSepharose column, the peak of siRNA-binding activity correlated well with that of the siRNA-generating activity. The gel shift assays were then performed in the presence of antibodies against Dcr-2 or R2D2. Both antibodies resulted in a supershift that was absent when their preimmune sera were used instead. This indicated that Dcr-2 and R2D2 are present in this siRNA-protein complex (Liu, 2003).
Purified Dcr-2, Dcr-2/R2D2, and Dcr-2/R2D2M recombinant proteins were examined for siRNA binding by the gel shift assay. Wild-type Dcr-2/R2D2 complex, but not Dcr-2 alone, binds to siRNA and produces a mobility shift indistinguishable from that of S100. The ability of the Dcr-2/R2D2 complex to bind siRNA is greatly diminished by point mutations within the two dsRNA-binding domains of R2D2. Thus, R2D2 is important for binding to the product (siRNA) rather than the substrate (dsRNA) of Dcr-2 (Liu, 2003).
Furthermore, when Dcr-2/R2D2 proteins and radiolabeled siRNA were exposed to ultraviolet (UV) light, both Dcr-2 and R2D2 were crosslinked to siRNA. This crosslinking was greatly diminished when the mutant Dcr-2/R2D2M complex was used instead, although similar amounts of proteins were used. Interestingly, two R2D2-siRNA crosslinked bands were observed, one at 45 kD and another at 52 kD: this probably represents R2D2 proteins covalently linked to one or two siRNA strands (the mass of each 21-nt siRNA strand was 7 kD). This was confirmed by the downshift of both R2D2-siRNA crosslinked bands to the original position of 38 kD His6-R2D2 protein after RNase treatment. These results indicate that Dcr-2 and R2D2 bind siRNA coordinately; this binding is dependent upon the dsRNA-binding domains of R2D2. On the basis of this finding, it was hypothesized that R2D2 might be involved in facilitating siRNA loading onto RISC. To test this hypothesis, the RISC activity was separated from the Dicer activity in S100 by polyethylene glycol (PEG) precipitation and then the partially purified RISC was combined with recombinant Dicer proteins to reconstitute the RNAi reaction. Although the majority of siRNA-generating activity was precipitated by 10% PEG, a substantial amount of RISC remained in the supernatant; this remaining material could be activated by addition of siRNA for sequence-specific mRNA degradation. The wild-type Dcr-2/R2D2 complex was much more effective than Dcr-2 alone or the mutant complex in promoting the dsRNA-initiated RISC activity in the 10% PEG supernatant. At 3 nM concentrations, wild-type Dcr-2/R2D2 stimulated the RISC activity by more than sevenfold, whereas Dcr-2 or Dcr-2/R2D2M stimulated only by twofold. Interestingly, the mutant complex blocked the RISC activity at 10 nM concentration, possibly as a result of dominant-negative effects. Furthermore, a similar phenomenon was observed in the siRNA-initiated RISC assay. Thus, the Dcr-2/R2D2 complex can enhance the siRNA- as well as the dsRNA-initiated RISC activities. This enhancement is not simply because of siRNA stabilization by Dcr-2/R2D2, as shown by comparing the stability of radiolabeled siRNA in RISC reactions (Liu, 2003).
To confirm that Dcr-2/R2D2 facilitates siRNA loading onto RISC, the association between AGO2, an essential component of RISC, and a 3'-biotinylated siRNA was followed by precipitation using streptavidin beads. The biotinylated siRNA was as active as unmodified siRNA in inducing RISC activities in S100. However, AGO2 protein was precipitated by streptavidin beads only when biotinylated siRNA was used, suggesting that interaction between AGO2 and siRNA was a specific interaction. RISC assays were then performed with the use of biotinylated siRNA in 10% PEG supernatant alone or in combination with recombinant Dcr-2, Dcr-2/R2D2, and Dcr-2/R2D2M proteins. Consistently, more AGO2 proteins were detected in the biotinylated siRNA precipitates when wild-type Dcr-2/R2D2 complex was used instead of Dcr-2 alone or the mutant complex. Together, these results indicate that Dcr-2/R2D2 not only generates siRNA from dsRNA but also binds to nascent siRNA and facilitates its loading onto RISC. The latter activity is dependent on the dsRNA-binding domains of R2D2 (Liu, 2003).
This model is supported by previous studies in which the direction of dsRNA processing was limited to one end of the dsRNA molecule. In these experiments, if dsRNA was processed from the 5' to 3' direction of the sense strand, it would generate RISC that can mediate degradation of the sense but not antisense target mRNA, and vice versa. However, if synthetic siRNA was used instead, it produced RISC that could degrade either sense or antisense target mRNA. Therefore, the newly synthesized symmetric siRNA product must not become a free molecule once it is generated from dsRNA. Rather, the nascent siRNA is likely to be retained by Dcr-2/R2D2 in a fixed orientation determined by the direction of dsRNA processing such that only the antisense strand can become the guiding RNA for RISC. Both R2D2 and RDE-4 contain tandem dsRNA-binding domains, interact with Dicer, and are required for the RNAi pathway. Thus, it is concluded that R2D2 is homologous to RDE-4. RDE-4 also interacts with RDE-1, an AGO2 homolog and a RISC component. Therefore, it is proposed that R2D2 and RDE-4 play a similar role in bridging the initiation and effector steps of the Drosophila and C. elegans RNAi pathways, respectively (Liu, 2003).
As a RNA helicase involved in posttranscriptional gene silencing, armitage mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).
Drosophila syncitial blastoderm embryo lysate has been used widely to study the RNAi pathway. However, armi flies lay few eggs, making it difficult to collect enough embryos to make lysate. To surmount this problem, lysates were prepared from ovaries manually dissected from wild-type or mutant females. Approximately 10 μl of lysate can be prepared from ~50 ovaries (Tomari, 2004).
The well-characterized siRNA-directed mRNA cleavage assay was used to evaluate the capacity of ovary lysate to support RNAi in vitro. Incubation in ovary lysate of a 5' 32P-cap-radiolabeled firefly luciferase mRNA target with a complementary siRNA duplex yielded the 5' cleavage product diagnostic of RNAi. siRNAs containing 5' hydroxyl groups are rapidly phosphorylated in vitro and in vivo, but modifications that block phosphorylation eliminate siRNA activity. Replacing the 5' hydroxyl of the antisense siRNA strand with a 5' methoxy group completely blocks RNAi in the ovary lysate. In Drosophila, siRNAs bearing a single 2'-deoxy nucleotide at the 5' end are poor substrates for the kinase that phosphorylates 5' hydroxy siRNAs (Nykanen, 2001). A comparison of initial cleavage rates shows that in ovary lysate, target cleavage was slower for siRNAs with a 2'-deoxy nucleotide at the 5' end of the antisense strand than for standard siRNAs. Furthermore, the rate of target cleavage was fastest when the siRNA was phosphorylated before its addition to the reaction. A similar enhancement from pre-phosphorylation was reported for siRNA injected into Drosophila embryos. It is concluded that lysates from Drosophila ovaries faithfully recapitulate RNAi directed by siRNA duplexes (Tomari, 2004).
Liu and colleagues have proposed that a heterodimeric complex, comprising Dicer-2 and the dsRNA binding protein R2D2, loads siRNA into RISC (Liu, 2003). Complex A contains the Dcr-2/R2D2 heterodimer. R2D2 and Dcr-2 are readily crosslinked to 32P-radiolabeled siRNA with UV light (Liu, 2003). An siRNA was synthesized containing a single photocrosslinkable nucleoside base (5-iodouracil) at position 20. The 32P-5-iodouracil siRNA was incubated with embryo lysate to assemble complexes, then irradiated with 302 nm light, which initiates protein-RNA crosslinking only at the 5-iodo-substituted nucleoside. Proteins covalently linked to the 32P-radiolabeled siRNA were resolved by SDS-PAGE. Two proteins -- ~200 kDa and ~40 kDa -- efficiently crosslinked to the siRNA. Both crosslinked proteins were coimmunoprecipitated with either α-Dcr-2 or α-R2D2 serum, but not with normal rabbit serum. Neither crosslink was observed in ovary lysates prepared from r2d2 homozygous mutant females, a result expected because Dcr-2 is unstable in the absence of R2D2 (Liu, 2003; Tomari, 2004).
The crosslinking was repeated, and the reaction analyzed by native gel electrophoresis to resolve complexes B, A, and RISC. Each complex was eluted from the gel and analyzed by SDS-PAGE. The R2D2 and Dcr-2 crosslinks were present in complexes A and RISC, but not B. In a parallel experiment, complexes B, A, and RISC were isolated (without crosslinking) and analyzed by Western blotting with either α-Dcr-2 or α-R2D2 antibodies. Again, complexes A and RISC, but not B, contained both Dcr-2 and R2D2. Finally, complex assembly was tested in ovary lysates prepared from r2d2 homozygous mutant females. Only complex B formed in these lysates. It is concluded that complex A contains the previously identified Dcr-2/R2D2 heterodimer (Liu, 2003, and that both Dcr-2 and R2D2 remain associated with at least a subpopulation of RISC, consistent with earlier reports that Dcr-2 in flies and both DCR-1 and the nematode homolog of R2D2, RDE-4, coimmunoprecipitate with Argonaute proteins (Tomari, 2004).
Native gel electrophoresis has been used to characterize complexes that mediate RNA interference (RNAi) in Drosophila. The data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. These results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 'holo-RISC' complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi (Pham, 2004).
Of these complexes, R1 forms the earliest. It does not require ATP to form and contains Dcr-2, which directly binds siRNAs. R1 corresponds to a previously identified 360 kDa siRNP complex that contains duplex siRNAs and is inactive for targeted mRNA cleavage (Nykanen, 2001). It is also probably equivalent to a dsRNA-processing complex isolated from Drosophila S2 cells that contains Dcr-2 and R2D2 (Liu, 2003). R1 shares many features with this complex and can also form in extracts prepared from S2 cells). Based on these results, as well as those of Liu (2003), it is concluded that the factors that form R1 process dsRNA and initiate RISC assembly. Furthermore, these results indicate that siRNAs must interact with Dcr-2 in order to reach RNAi effector complexes and that Dcr-2/siRNA association within the R1 complex is the first detectable step in the RISC-assembly pathway. Intriguingly, UV crosslinking results indicate that Dcr-2 can weakly or transiently bind 5'-hydroxyl-bearing siRNAs in the R1 complex. Nykanen (2001) proposed that the siRNA 5'-phosphate serves a 'licensing' function in the RNAi pathway, allowing the silencing machinery to recognize bona fide siRNAs; if this is true, these results indicate that siRNA 5'-phosphorylation status is monitored within the R1 complex after the initial Dcr-2/siRNA interaction has occurred. This could be achieved by fast dissociation of 5'-hydroxyl siRNAs from the Dcr-2 complex (Pham, 2004).
The R2 complex has not yet been characterized in detail because it neither survives gel filtration chromatography nor sediments cleanly in a sucrose gradient. For now, it is only possible to speculate about its composition and role in RNAi. Based on its kinetics and behavior in a chase experiment, R2 may be an intermediate that links R1 and R3, although it is not certain whether it is 'on pathway.' Since R2 forms so rapidly in this experiment, it might arise from R1 binding to a set of pre-associated factors (or to a single factor). This binding may then trigger the recruitment of still more factors, possibly accompanied by conformational rearrangements, because relative to the other complexes, R2's mobility in a native gel decreases slightly with time. siRNA unwinding may occur during this process, leading to formation of R3 (Pham, 2004).
The R3 complex is significantly enhanced by incubation with ATP, a known requirement for activated RISC formation. R3 is very large (80S) and cofractionates with the known RISC-associated components VIG, TSN, Ago2, and dFXR. The siRNAs associated with R3 are single stranded, and the complex co-sediments precisely with mRNA target cleavage activity. Finally, R3 can bind a cognate mRNA target. Based on these results, it is concluded that R3 is an RNAi effector complex that is significantly larger than those previously described (Pham, 2004).
The experiments suggest that R3 is a ribosome bound silencing complex. Indeed, several lines of evidence link RNAi to translation and therefore support this possibility. In Drosophila oocytes, untranslated mRNAs are refractory to RNAi, whereas those actively undergoing translation are not (Kennerdell, 2002), indicating that mRNAs most susceptible to RNAi are those that can also interact with the translational machinery. MicroRNAs have been identified within RISC (Hutvagner, 2002) and can also affect protein synthesis from target messages (Olsen, 1999), suggesting further functional links between RISC and ribosomes. In S2 cells, Drosophila RISC components pellet with ribosomes and other large complexes after high-speed centrifugation (Hammond, 2001; Caudy, 2002, 2003). Two of these components, Ago2 and dFXR, are found in complexes that include 5S rRNA as well as two ribosomal proteins, L5 and L11 (Ishizuka, 2002). siRNAs have also been found in association with the translational machinery (Djikeng, 2003), interacting with polyribosomes in the protozoan Trypanosoma brucei (Pham, 2004 and references therein).
If R3 is a ribosome bound complex, then it is likely that RISC binds during or after siRNA unwinding and activation. This assertion is based both on the apparent ATP dependence of R3 formation and on the preponderance of single-stranded siRNAs within the complex. It has been shown that siRNA unwinding requires ATP (Nykanen, 2001). Although the possibility cannot be rule out that a potential RISC/ribosome interaction requires ATP for unrelated reasons, the data indicate that siRNA unwinding is one source of that requirement. Once formed, the R3 complex may be biochemically separable since smaller, active RISCs have been identified (Hammond, 2001; Nykanen, 2001; Martinez, 2002). If so, then R3 may represent a holo-silencing complex that includes factors not absolutely required for mRNA cleavage activity in vitro. These factors may include regulatory elements or those involved in the miRNA silencing pathway (Pham, 2004).
Because dsRNA processing and mRNA target cleavage activities are biochemically separable, Dicer is generally thought to release siRNAs to RISC, playing no apparent role in the effector phase of RNAi. However, the presence of Dcr-2 and R2D2 in R3-containing sucrose gradient fractions raises interesting questions about their role in RNA silencing, especially since they are not detectable in the corresponding fractions collected from naive extracts. Liu (2003) has argued that the Dcr-2-associated protein R2D2 conveys siRNAs from the RNAi initiator complex to the effector complex. The current data instead indicate that the Dcr-2/R2D2 initiator complex, itself, is incorporated into the effector in a RISC assembly pathway (Pham, 2004).
Sucrose gradient experiments indicate that RISC components may associate with each other even in the absence of exogenous siRNAs. This association is not likely to be an apo-complex because fractionated naive extracts cannot form R3 when they are incubated with siRNA duplexes after fractionation. Rather, it may represent endogenous microRNA-programmed RISC complexes or perhaps a partially preformed 'holo-RISC' that lacks essential assembly factors (such as Dcr-2 and R2D2). Since the silencing activities of RISC and the miRNP are apparently governed by the degree of complementarity between trigger and target, and not by the identity of the silencing complex (Hutvagner, 2002; Doench, 2003; Zeng, 2003), R3 may be able to form on both siRNAs and miRNAs. If so, then there may be distinct mechanisms for incorporating siRNA and miRNA triggers into the silencing complex. siRNA incorporation into RISC is likely to proceed on a Dcr-2/R2D2-dependent pathway since Dcr-2 and R2D2 can process dsRNAs (Liu, 2003) but are not detectable in sucrose gradient fractions collected from naive extracts. However, miRNA incorporation may proceed on a different pathway to the silencing complex, possibly relying on Dcr-1. There is support for this model in the apparent division of labor between Dcr-1 and Dcr-2 in processing miRNA and dsRNA triggers (Lee, 2004). If this model is correct, then the observed size of R3 may reflect the diverse roles it may play, from mRNA cleavage to translational control (Pham, 2004).
Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer, alone, can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex (RLC), which also requires Ago2 for assembly into RISC. R2 was compared to the RLC and the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. The requirements for RISC formation were examined; the siRNA 5' termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2' position of the 5'-terminal nucleotide also affects RISC assembly because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference (RNAi) pathway (Pham, 2005).
To act as silencing triggers, double-stranded siRNAs must be channeled through an ordered RISC assembly pathway that results in the selection of one strand and the destruction of the other. The results indicate that siRNA ends are recognized at multiple steps in the pathway and that these recognition events determine whether the siRNAs will become incorporated into the RISC or blocked from further assembly. The first recognition event occurs at initiation, when Dcr-2 and R2D2 bind the siRNA to form the RDI complex. RDI formation requires 5'-phosphorylated siRNA; however, the siRNA need not be phosphorylated on both strands. siRNAs bearing a single 5'-phosphate group are ineffective at guiding mRNA cleavage. This is true for siRNAs phosphorylated only on the guiding strand as well as those phosphorylated only on the nonguiding strand. To explain these observations, a model has been involked in which R2D2 acts as a sensor for siRNA asymmetry. It has been suggest that a 5' phosphate on the thermodynamically disfavored 'passenger' strand is required for stable R2D2 binding, facilitating siRNA incorporation into the RLC and the RISC (Pham, 2005 and references therein).
When only one of two siRNA strands is phosphorylated, R2D2 and Dcr-2 can still avidly engage the siRNA, probably with R2D2 located at the phosphorylated end as predicted. Despite this, singly phosphorylated siRNA duplexes are still ineffective silencing triggers. The data indicate that a second 5'-recognition event occurs after R2D2 binding. RNA unwinding and activation can proceed, but only if the siRNA bears the second 5' phosphate at the end not occupied by R2D2. Since Ago2 is required both for siRNA unwinding and target mRNA cleavage, it may be the factor that recognizes the second 5' phosphate. An archaebacterial PIWI protein has been shown to have conserved residues that interact with the 5'-phosphate group of siRNA-like duplexes. Furthermore, when analogous residues were mutated in the human Ago2 PIWI domain, target mRNA cleavage is compromised. These results seem to implicate Ago2 in the second 5' recognition event. Alternatively, Ago2 may not engage the 5' phosphate until later in the assembly pathway. In this case, some other factor may interact with the second 5' phosphate upstream of Ago2, causing the RISC assembly defect that is observed with singly phosphorylated siRNA duplexes (Pham, 2005).
Like the 5'-phosphate group, the 2' hydroxyl of the 5'-terminal nucleotide can influence RISC assembly. A 5'-terminal 2'- deoxyribose modification on one siRNA strand inhibits the cognate strand from entering the RISC. Since the unmodified, cognate strand does not accumulate in an unwinding assay, it is probably degraded. In contrast, the modified strand has enhanced activity and is apparently favored for entry into the RISC. There are at least two (non-mutually exclusive) explanations for these observations. The 2'-deoxyribose may be a negative determinant for R2D2 binding. In support of this possibility, the dT-PO4-modified Pp-luc-switch siRNA forms very little of the RDI and R2 complexes, even though it has a relatively stable and phosphorylated terminus that R2D2 would be expected to bind. Decreased R2D2 binding would bias the 'sensor' of siRNA asymmetry, leading to increased selection of the modified strand and increased degradation of the unmodified strand, as is observed. Alternatively, the 2'-deoxyribose-modified strand may have an increased affinity for some downstream RNAi factor, thereby enhancing its entry into the RISC. This may also explain why a single-stranded dTPO4-modified siRNA is better at guiding target mRNA cleavage than the analogous rU-PO4 control. In either case, the observations are surprising because the RNAi apparatus generally tolerates limited modifications at the 2' position of siRNA duplexes, both on the sense and antisense strands. However, completely substituting siRNAs with 2'-deoxy modifications on either the sense or antisense strand abolishes their ability to guide target mRNA cleavage, perhaps by altering the helical geometry of the duplex. Since there does not seem to be a general requirement for 2' hydroxyls along the length of the siRNA nonguiding strand, the defect observed for 5'- terminal 2'-deoxyribose modifications could reflect a specific recognition event that cannot yet be fully defined (Pham, 2005).
Since a 5'-terminal 2'-deoxyribose modification on one siRNA strand can inhibit the activity of the cognate strand, it should reduce the off-target effects that can complicate RNAi-mediated gene silencing. Thus, this simple and inexpensive modification should be considered in the design of effective siRNA reagents for targeted gene knock-down (Pham, 2005).
Finally, progress has been made in the goal of developing a more unified understanding of RISC assembly in Drosophila. The R2D2/Dcr-2 heterodimer, alone, can initiate RISC assembly by binding siRNAs to form the RDI complex. Like the RDI, Complex B has been proposed to act at initiation. Since this complex can form in the absence of R2D2, it is upstream of the RDI and may play an auxiliary role in RISC assembly. Complex B may transfer bound siRNA directly to the RDI in addition to (or instead of) the RLC. Continuing investigation will be required to resolve this issue (Pham, 2005).
The results suggest that the RLC and R2 complexes have more in common than previously thought. Based on side-by-side comparisons, both complexes can form in extracts that lack Ago2 and in extracts depleted of ATP. Furthermore, both complexes can assemble on siRNAs that lack a phosphate on one of the two siRNA strands. Based on these observations, R2 and the RLC are probably identical complexes, and R2 is now referred to as the RLC (Pham, 2005).
Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. This study used a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro (Preall, 2006).
In Drosophila, Dcr enzymes are required for RISC assembly as well as dsRNA processing, suggesting that the two phases of RNAi might be functionally coupled in a manner that affects siRNA strand selection. However, these experiments indicate that Drosophila RISC assembly and siRNA strand selection are not significantly influenced by the dsRNA processing step and that the thermodynamic asymmetry rules apply equally well with processed and unprocessed siRNAs in this system. This suggests that Drosophila Dcr enzymes do not channel newly generated siRNAs directly into RISC, but rather release the siRNAs into solution (or onto another factor) before they enter the RISC assembly pathway (Preall, 2006).
Several observations have suggested that thermodynamic asymmetry governs strand selection for processed RNAi triggers. MicroRNAs (miRNAs) are diced from stem-loop precursors, and in most instances only one strand of the processed miRNA duplex is stably incorporated into RISC. The mature strand can be present at either the 5' or the 3' end of the stem-loop, but either way, the selected strand is generally compatible with the thermodynamic asymmetry guidelines. In addition, artificial dsRNAs introduced into plant cells give rise to a stable set of siRNAs that adhere to the asymmetry rules. Similar results have been reported with natural dsRNAs in plants. However, interpretation of these results is difficult because the Dcr processing polarities were not defined, and it is also not clear whether small RNA stability is always a suitable surrogate measure of RISC assembly. Furthermore, plant cells (unlike insect and mammalian cells) export siRNAs into the vasculature to enable systemic RNAi, and therefore the plant dsRNA processing machinery may have specifically evolved the propensity to release newly processed siRNAs. Thus the applicability of the plant analyses to insects and mammals has not been clear (Preall, 2006).
While this work was in progress, Rose (2005) characterized modified ~27 nt duplexes that force a defined Dcr processing polarity and give rise to specific, predictable 21 nt siRNAs. Experiments with these Dcr substrate RNAs revealed that hDcr processing polarity can in fact influence siRNA strand selection in transfected human cells, although it does not completely supercede thermodynamic asymmetry. The reasons for the discrepancy between the results and those of Rose are not clear, although one possibility is that different Dcr enzymes may vary in their tendencies to remain associated with newly generated siRNAs. It is noteworthy that Drosophila Dcr-2 (which is primarily devoted to the siRNA pathway) appears to lack the canonical PAZ domain that normally provides Dcr with a binding pocket for 2 nt 3' overhangs. A PAZ domain is present in hDcr, and mutational analyses indicate that the hDcr PAZ domain assists with dsRNA binding and processing when a 2 nt 3' overhang is present. The apparent lack of a PAZ domain in Dcr-2 may compromise its ability to remain bound to newly cleaved siRNA. It is curious that Drosophila Dcrs are required for RISC assembly but do not appear to couple dsRNA processing to siRNA strand selection, whereas mammalian Dcrs are not required for RISC assembly but apparently do couple dsRNA processing to siRNA strand selection (Preall, 2006).
Finally, it remains to be determined whether Dcr enzymes associate with long dsRNA processing substrates and siRNA RISC-assembly substrates in the same way. This issue is undoubtedly important for understanding the functional relationship between Dcr's roles in the initiator and effector phases of RNAi. Crystal structures of E. coli RNase III, an ancestor of eukaryotic Dcrs, are likely to be informative. The structural data, and models derived from them, depict a protein that can engage dsRNAs in a dynamic fashion. A single dsRBD on each subunit of the RNase III homodimer is tethered to the endonuclease domain by a flexible linker that can rotate roughly 90° around the catalytic core. Thus, there are likely to be at least two binding modes for dsRNA in complex with an RNase III enzyme: one in which the dsRBD braces the RNA helix from either side as it is channeled into the catalytic cleft, and another where the dsRBD holds the dsRNA above and orthogonal to the active site. It is possible that Dcr enzymes also exhibit alternate dsRNA binding modes depending on whether they are actively processing dsRNA or channeling siRNA into RISC. Interconversion between these two conformations may require at least transient release of the siRNA product. Additional dynamic dsRNA/protein interactions during dsRNA processing and RISC assembly presumably involve the dsRNA binding proteins Loquacious/R3D1, R2D2, and TRBP, which associate with Dcr-1, Dcr-2, and hDcr, respectively. Further functional analysis of Dcr's PAZ, RNase III, and dsRNA binding domains, aided by recent advances in the structural biology of Dcr, will be necessary to understand Dcr's roles in the transition between the initiation and effector phases of RNAi (Preall, 2006).
Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, that arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. This study shows that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2. Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles (Czech, 2008).
Drosophila expresses five Argonaute proteins, which segregate into two classes. The Piwi proteins (Piwi, Aubergine and AGO3) are expressed in gonadal tissues and act with Piwi-interacting RNAs (piRNAs) to suppress mobile genetic elements. The Argonaute class contains AGO1 and AGO2. AGO1 binds microRNAs (miRNAs) and regulates gene expression. The endogenous binding partners of AGO2 have remained enigmatic (Czech, 2008).
Transgenic flies were generated expressing epitope-tagged AGO2 under the control of its endogenous promoter. Tagged AGO2 localizes to the cytoplasm of germline and somatic cells of the ovary. Immunoprecipitated AGO2-associated RNAs differ in their mobility from those bound to AGO1. Deep sequencing of small RNAs from AGO1 and AGO2 complexes yielded 2,094,408 AGO1-associated RNAs and 916,834 AGO2-associated RNAs from Schneider (S2) cells, and 455,227 AGO2-associated RNAs from ovaries that matched perfectly to the Drosophila genome. Three libraries were sequenced derived from 18-29-nucleotide RNAs (936,833 sequences from wild-type ovaries, 1,042,617 sequences from Dicer-2 (Dcr-2) mutant ovaries, and 1,946,339 sequences from loquacious (loqs) mutant ovaries) and an 18-24-nucleotide library from wild-type testes (522,848 sequences). Finally, the analysis included 92,363 published sequences derived from 19-26-nucleotide RNAs from S2 cells (Czech, 2008).
Among the ~50% of AGO2-associated RNAs from S2 cells that did not match the genome, ~17% matched the flock house virus (FHV), a pathogenic RNA virus and reported target for RNAi in flies. These probably arose because of persistent infection of the S2 cultures (Czech, 2008).
After excluding presumed degradation products of abundant cellular RNAs, each of the total RNA libraries were divided into two categories: annotated miRNAs and the remainder. For the S2 cell library, the size distribution of these populations formed two peaks, with non-miRNAs lying at 21 nucleotides and miRNAs exhibiting a broader peak from 21 to 23 nucleotides. Libraries derived from AGO1 and AGO2 complexes almost precisely mirrored these two size classes. In the ovary library, this approach revealed three size classes. Whereas two reflected those seen in S2 cells, a third class comprised piRNAs. Again, RNA size profiles from AGO2 or Piwi family immunoprecipitates mirrored those within the total ovary library. These data demonstrate that AGO2 is complexed with a previously uncharacterized population of small RNAs (Czech, 2008).
Whereas known miRNAs comprised more than 97% of AGO1-associated RNAs in S2 cells, they made up only 8% or 20% of the AGO2-bound species in S2 cells or ovaries, respectively. The remaining small RNAs in AGO2 complexes formed a complex mixture of endogenous siRNAs (endo-siRNAs). Among these, transposons and satellite repeats contributed substantially to AGO2-associated small RNAs in S2 cells (27%) and ovaries (53%). The nature of the transposons giving rise to abundant siRNAs in ovaries and S2 cells differed substantially, probably reflecting differential expression of specific transposons in these tissues. Unlike piRNAs, neither somatic nor germline siRNAs exhibited a pronounced enrichment for sense or antisense species (Czech, 2008).
In accord with these findings, knockdown of AGO2 in S2 cells leads to increased expression of several mobile elements. In the germ line, the Piwi-piRNA system has been reported as the dominant transposon-silencing pathway. Nevertheless, several transposons, with a potential to be targeted by siRNAs, were substantially derepressed in AGO2 mutant or Dcr-2 mutant ovaries. Although comparisons of relative abundance were difficult, both piRNAs and siRNAs mapped to piRNA clusters, with the regions that generate uniquely mapping species generally overlapping. Thus, piRNA loci are a possible source for antisense RNAs matching transposons and might serve a dual function in small RNA generation. Considered together, these data suggest that endo-siRNAs repress the expression of mobile elements, in some tissues acting alongside piRNA pathways (Czech, 2008).
To probe the nature of the remaining endo-siRNAs, genomic sites, which give rise to multiple uniquely mapping RNAs that do not fall into heterochromatic regions, were computationally extracted. These generally segregated into two categories, termed structured loci or convergently transcribed loci (Czech, 2008).
Transcripts from structured loci can fold to form extensive double-stranded RNA directly. The two major loci, termed esi-1 and esi-2, gave rise to half of the 20 most abundant endo-siRNAs in ovaries and also generated siRNAs in embryos, larvae and adults. esi-1, annotated as CG18854, can produce an ~400-base pair (bp) dsRNA through interaction of its 5' and 3' untranslated regions. esi-2 overlaps with CG4068 and consists of 20 palindromic ~260-nucleotide repeats. All siRNAs derived from these two loci arise from one genomic strand. In some previously characterized instances (for example, Arabidopsis trans-acting-siRNAs) Dicer generates 'phased' siRNAs with 5' ends showing a 21-nucleotide periodicity. In all tissues examined, esi-1 and esi-2 produced phased siRNAs, consistent with a defined initiation site for Dicer processing. Phasing was not observed for viral or repeat-derived siRNAs. Finally, siRNAs from both loci also joined AGO1 in proportions greater than siRNAs produced from transposons and repeats, perhaps owing to the imperfect nature of the dsRNA that they produce (Czech, 2008).
AGO2 regulates gene expression by cleavage of complementary sites rather than by recognition of seed sites typical of AGO1-miRNA-mediated regulation. Possible targets of endo-siRNAs were sought by identifying transcripts with substantial complementarity. A highly abundant siRNA from esi-2 is highly complementary to the coding sequence of the DNA-damage-response gene mutagen-sensitive 308 (mus308). Using a modified rapid amplification of cDNA ends (RACE) protocol, mus308 fragments were detected with 5' ends corresponding precisely to predicted endo-siRNA cleavage sites. Moreover, AGO2 and Dcr-2 loss consistently increased mus308 expression in testis and to a lesser extent in ovaries, consistent with the relative abundance of esi-2 siRNAs in these tissues. Finally, a reporter gene containing two mus308 target sites was significantly derepressed in S2 cells on depletion of Dcr-2 or AGO2 but not of Dcr-1 or AGO1. Although extensive complementarity between other endo-siRNAs and messenger RNAs was rare, several esi-1-derived siRNAs complementary to CG8289 were found, suggesting a potential regulatory interaction in vivo (Czech, 2008).
A second group of siRNA-generating loci contained regions in which dsRNAs can arise from convergent transcription. If sorted for siRNA density, most of the top 50 ovarian and S2 cell siRNA loci lay in regions where annotated 3' UTRs or expressed-sequence-tags corresponding to convergently transcribed protein-coding genes overlap. Typically, siRNAs arise on both genomic strands but only from overlapping portions of convergent transcripts. Examining all 998 convergently transcribed gene pairs in the Drosophila genome with annotated overlapping transcripts, the peak abundance of ovarian siRNAs was found to be at the centre of the overlap, with sharp declines away from this region. In an alternative arrangement, Pgant35A produces sense and antisense siRNAs across its entire annotated transcript, consistent with expressed-sequence-tag support for antisense transcription traversing this locus (Czech, 2008).
Thus, a large number of Drosophila genes generate endogenous siRNAs, with most having perfect complementarity to the 3' UTRs of neighbouring genes. Relative levels of endo-siRNAs generated from each convergent transcription unit were low, and no or little change (up to a ~1.3-fold increase) was found in the expression of such genes in AGO2 mutant ovaries. Possibly, the level of small RNAs produced by this genomic arrangement is inconsequential, amounting to noise within silencing pathways. However, there are probably circumstances wherein regulation by such arrangements might substantially impact expression (Czech, 2008).
In S2 cells, two neighbouring loci encoded nearly 16% of AGO2-associated RNAs. These reside within a large intron of klarsicht and did not generate siRNAs in any other tissue. A similar locus, corresponding to CG14033, was found within an intron of thickveins and gave rise to testis-specific siRNAs. Although the function of both siRNA clusters is unclear, the thickveins cluster shares considerable complementarity to CG9203, and loss of AGO2 and Dcr-2 mildly increased CG9203 mRNA levels in testis but not in ovaries (Czech, 2008).
Dcr-2 has been implicated in the production of siRNAs from viral replication intermediates or exogenously introduced dsRNAs, whereas Dcr-1 has been linked to miRNA biogenesis. In agreement with these observations, all endo-siRNA classes were lost in Dcr-2 mutant ovaries. To obtain more insight into the genetic requirements for endo-siRNA biogenesis and stability, components of siRNA and miRNA pathways were depleted in S2 cells, and levels of abundant siRNAs derived from structured loci were analysed . Although depletion of Dcr-2 and AGO2 resulted in substantial reductions in siRNA levels, little or no changes were observed on Drosha, Pasha, Dcr-1 or AGO1 depletion. Unexpectedly, virtually no requirement was found for the Dcr-2 partner R2D2 but a strong requirement was found for the Dcr-1 partner Loquacious. Only one analysed siRNA exhibited partial dependence on R2D2, potentially correlating with the extensive dsRNA character of its precursor duplex. Artificial sensors for endo-siRNAs from esi-1 and esi-2 in S2 cells gave patterns of de-repression that matched analysis of endo-siRNA levels (Czech, 2008).
Analysis of the most abundant siRNA from esi-2 in flies mutant for Dcr-2, AGO2, r2d2 or loqs extended these findings from cell culture. To examine the unexpected requirement for loqs more broadly, small RNAs were sequenced from loqs-mutant ovaries and a near complete loss of endo-siRNAs from structured loci was observed. A much smaller impact of loqs was seen on endo-siRNAs derived from repeats and convergent transcription units. However, an involvement of Loqs and not R2D2 in the function of siRNAs derived from perfect dsRNA precursors was supported by analysing the impact of depleting siRNA/miRNA pathway components on the ability to suppress FHV replication in infected S2 cell cultures (Czech, 2008).
The results uncover an unanticipated role for Loqs in siRNA biogenesis and suggest that R2D2 has a lesser impact on at least two types of endogenous siRNAs. It is well established that Loqs partners with Dcr-1 for miRNA processing. To probe a molecular interaction with Dcr-2, Loqs binding partners were analyzed using quantitative proteomics. Dcr-1 and Dcr-2 were both abundant in Loqs immunoprecipitates from cultured cells and flies, supporting a physical interaction between Dcr-2 and Loqs (Czech, 2008).
Among animals, endo-siRNA pathways have so far been restricted to Caenorhabditis elegans. The current results extend the prevalence of such systems to Drosophila and parallel recent discoveries of an endo-siRNA pathway in mouse oocytes. These systems have many common features but also key differences. In both, siRNAs collaborate with piRNAs to repress transposons. Also, mouse and Drosophila both generate endo-siRNAs from structured loci. In mouse, dsRNAs can form by pairing of sense protein-coding transcripts with antisense transcripts from pseudogenes. Whether or not transcripts from unlinked sites lead to siRNA production in Drosophila is unclear. However, transposon sense transcripts may hybridize to antisense sequences transcribed from piRNA clusters to form endo-siRNA precursors. In flies, a much larger number of genic loci enter the pathway as compared to mice because convergent transcription of neighbouring genes frequently creates overlapping transcripts. Overall, annotation of the Drosophila genome indicates that a significant proportion is transcribed in both orientations, providing widespread potential for dsRNA formation. This property is shared by many other annotated genomes, raising the possibility that the RNAi pathway has broad impacts on gene regulation. Viewed in combination, these studies suggest an evolutionarily widespread adoption of dsRNAs as regulatory molecules, a property previously ascribed only to miRNAs (Czech, 2008).
Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, flies were constructed expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners (Fukunaga, 2012).
The simplest interpretation of the data that Loqs-PA and Loqs-PB both decrease the KM of Dcr-1 for pre-miRNA substrates is that Loqs partner proteins increase the affinity of Dcr-1 for some pre-miRNA substrates. For other pre-miRNAs, Loqs-PB can also increase Dcr-1 enzyme turnover. The functions of Dicer partner proteins in small RNA biogenesis in flies is summarized as follows: preventing Dcr-2 from processing pre-miRNAs (R2D2), promoting the processing of 'foreign' dsRNA and of esiRNAs and other endo-siRNA precursors (Loqs-PD), enhancing the efficiency of pre-miRNA processing by Dcr-1 (Loqs-PA and PB), and tuning cleavage site choice by Dcr-1 for a small number of pre-miRNAs (Loqs-PB) (Fukunaga, 2012).
The last two functions are surprising because the number of pre-miRNAs that require Loqs-PB and not Loqs-PA for the efficient production of their miRNAs is small and even fewer require Loqs-PB to produce the correct isomir. Nonetheless, the results suggest that the loss of GSCs in flies lacking Loqs- PB reflects the reduced abundance of specific miRNAs such as miR-79, miR-283, miR-305, miR-311, and miR-318 or perhaps the production of the wrong isomirs for miR-307a, miR-87, or miR-316. Among these, miR-318 is the most abundant miRNA in ovaries (140,000 ppm in w1118 ovaries but only 8 ppm in heads). Future experiments using Loqs-independent versions of pre-miR-318 and other miRNAs should help test this idea (Fukunaga, 2012).
The amino acid sequence of Loqs-PB suggests it is slightly more related to PACT (28% identity; 34% similarity) than to TRBP (24% identity; 33% similarity). Nonetheless, our data suggest that the functional homolog of Loqs-PB in mammals is TRBP. Like Loqs-PA and Loqs-PB, TRBP may act generally to enhance the binding of Dicer to pre-miRNA. Such a role for TRBP is consistent with earlier observations that the two cleavage reactions catalyzed by mammalian Dicer are less well coordinated in the absence of TRBP. But like Loqs-PB, TRBP also helps Dicer produce specific isoforms of a one or more miRNAs. This novel function of Dicer partner proteins may be widely conserved, enabling plants and animals to effectively and accurately dice difficult but important pre-miRNA substrates. Perhaps the Drosha-binding partner Pasha in flies or DGCR8 in mammals similarly tunes pri-miRNA cleavage site choice by Drosha (Fukunaga, 2012).
The stem, but not the loop, of pre-miR-307a enables Loqs-PB to influence where Dcr-1 cleaves. One possible explanation for how Loqs-PB and TRBP change where Dcr-1 and Dicer cleave is that the mismatches and internal loop causes the stems of pre-miR-307a, pre-miR-87, and mammalian pre-miR-132 to be longer than the corresponding A-form helix; binding of Loqs-PB or TRBP might then 'shrink' the stems. In contrast, Loqs-PB binding may extend of the stem of premiR-316. Because Loqs-PB does not change where Dcr-2 cleaves pre-miR-307a, Loqs-PB likely acts only when bound to Dcr-1 (Fukunaga, 2012).
The data suggest that the effect of Loqs-PB is biologically relevant. The long miR-307a isomir predominates in wild-type flies, and miR-307a23-mer but not miR-307a21-mer can repress the Gk and tara mRNAs in vivo. Why has evolution failed to select for easier-to-dice variants of pre-miR-307a and premiR-87 in flies and pre-miR-132 in mice, so that a specific partner is no longer needed to ensure production of the 'right' isomir? The persistence of the miR-307a21-mer in wild-type flies, for example, suggests that most, if not all, abundant isomirs have distinct biological functions and that the optimal function of miRNAs like miR-307a, miR-87, and miR-132 requires a defined ratio of isomirs (Fukunaga, 2012).
Although no altered regulation of predicted miR-307a21-mer targets was detected in ovaries from flies lacking Loqs-PB, miR-307a21-mer may have functions in other tissues or times in development. In flies, mice, and humans, the relative abundance of miRNA isomirs generated by different Dicer cleavage sites, including 50 isomirs with distinct seed sequences, varies among tissues and developmental stages. Perhaps the abundance of Loqs-PB versus Loqs-PA or TRBP versus PACT is regulated across development and differentiation to ensure the correct relative abundance of isomirs from various pre-miRNAs, much as the ratios of alternatively spliced mRNAs are regulated in different tissues and cell types. loqsKO flies rescued with transgenes producing individual Loqs isoforms should facilitate the testing of this idea in vivo (Fukunaga, 2012).
In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. A previous study has shown that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. This study reports that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5' terminal phosphate and a two-nucleotide, 3' overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. It is proposed that inorganic phosphate occupies a PAZ domain pocket required to bind the 5' terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. This study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme (Fukunaga, 2014).
Screens of three major autosomal arms of Drosophila have identified more than 15 loci that when mutated result in stronger pigmentation in a GMR-wIR background. One such locus, identified in the screen of the right arm of the second chromosome, was a homozygous viable complementation group consisting of 39 alleles. Noncomplementation was based on a strongly suppressed eye color phenotype in the presence of GMR-wIR. To genetically map the locus, Drosophila single nucleotide polymorphism (SNP) markers were used. Mapping placed the locus within a 568 kb interval of 54C, an interval that contains a Dicer gene. Drosophila contains two genes in the Dicer family, dicer-1 and dicer-2 (Lee, 2004).
The dcr-2 gene is located within the interval that contained the complementation group of suppressor mutations. The dcr-2 gene was sequenced in six independent mutants, and each mutant had base changes that significantly altered the predicted protein product. Two alleles contained premature stop codons that would produce truncated proteins, lacking the RNase III domains essential for dsRNA processing activity. These two mutations likely represent null alleles of dcr-2. To confirm that the mutant complementation group corresponded to dcr-2, transformation rescue was performed with a 7.2 kb genomic fragment that spans the dcr-2 transcription unit. This fragment completely rescued the phenotype associated with a homozygous dcr-2 null mutation (Lee, 2004).
It was next asked if the phenotype associated with dcr-2 mutants resulted from a defect in dsRNA processing. The levels of siRNAs generated from GMR-wIR were examined in the eyes of wild-type and dcr-2 mutants. The dcr-2 null mutants exhibit a large reduction in siRNA levels when compared to wild-type. This reduction did not result from instability or low-level expression of GMR-wIR dsRNA, since GMR-wIR precursor RNAs were present at levels comparable to wild-type. These data indicate that Dcr-2 plays a major role in dsRNA processing. Interestingly, substitution mutants in the Dcr-2 helicase domain were as impaired for siRNA production as null mutants. One of these, the dcr-2G31R mutant, changes one of the invariant GXGXXG residues in the ATP binding site of the helicase domain. Thus, Dcr-2 requires a functional helicase domain for dsRNA processing (Lee, 2004).
Flies homozygous for null dcr-2 alleles are viable and fertile, and are morphologically normal in external appearance. Since miRNAs are indispensable for growth and development in Drosophila, the dcr-2 phenotype suggests that Dcr-2 is not essential for pre-miRNA processing. To address this, levels of the miRNA let-7 were examined in dcr-2 null mutants. The dcr-2 mutants exhibited mature let-7 RNA levels comparable to those of wild-type controls. This confirms that Dcr-2 is not required for the processing of pre-miRNAs (Lee, 2004).
Previous work showed that RNAi is established in the Drosophila female germline (Kennerdell, 2002). To examine whether Dcr-2 is required for mRNA degradation in eggs, dcr-2 mutant eggs were injected with dsRNA corresponding to the bicoid gene, which is maternally expressed. Subsequently, bicoid mRNA levels were assayed by RT-PCR. Wild-type eggs displayed rapid reduction in bicoid transcript abundance after dsRNA injection. In contrast, dcr-2 null mutant eggs showed no significant reduction in bicoid transcript abundance, indicating that dcr-2 is required for effective RNAi in the female germline. A similar effect was observed in dcr-2 mutants bearing substitutions in the Dcr-2 helicase domain (Lee, 2004).
It was next asked whether the RNAi defect in dcr-2 eggs is simply due to defective siRNA production. To test this hypothesis, eggs were injected with a synthetic siRNA corresponding to the bicoid gene and subsequently bicoid transcript levels were assayed. Wild-type eggs exhibited loss of bicoid mRNA in response to siRNA injection. In contrast, dcr-2 null mutant eggs exhibited an impaired RNAi response to siRNA. Five-fold more bicoid mRNA was present in dcr-2 mutant eggs compared to wild-type eggs after siRNA treatment. This result indicates that Dcr-2 also functions downstream of siRNA production in the RNAi pathway. Interestingly, substitution mutants in the Dcr-2 helicase domain were unimpaired for siRNA-dependent RNAi, suggesting that a functional helicase activity is not required for Dcr-2 to mediate its downstream function (Lee, 2004).
These experiments established an important though not absolute role for Dcr-2 in Drosophila RNAi. Since another Dicer (Dcr-1) is present in Drosophila, it is possible that it has a redundant function with Dcr-2. The dcr-1 gene is located at 94C4 on the third chromosome, and a mutation was found that mapped by linkage and complementation analysis to that region. The coding sequence of dcr-1 in the mutant contained a premature stop codon such that the truncated product lacks the PAZ and RNase III domains. Moreover, dcr-1 mRNA is not detectable in the mutant as determined by RT-PCR and Northern blot analysis, suggesting that the transcript is unstable when truncated Dcr-1 protein is produced. This transcript null dcr-1Q1147X mutant exhibited an eye color phenotype when assayed in a GMR-wIR genetic background. The mutant appeared to partially suppress silencing by GMR-wIR, with patches of dark orange eye color. In addition, the eye was half its normal size, the organization of ommatidial facets was disrupted, and sensory bristles were missing over the eye surface. Other bristles, which flank the eye surface, were sometimes absent or exhibited hyperplasia (Lee, 2004).
Despite an effect on white gene silencing, the dcr-1 mutant had normal levels of wIR siRNAs. This observation is consistent with Dcr-2 processing the great majority of wIR dsRNA. It is further consistent with a central role for Dicer helicase activity in dsRNA processing, since Dcr-1 lacks a DExH-box helicase domain. If the dcr-1 mutant has normal dsRNA processing, why is it partially disrupted for gene silencing? To answer this, clones of homozygous dcr-1 mutant germ cells were generated in heterozygous females, and then dcr-1 mutant eggs were injected with either dsRNA or siRNA complementary to bicoid transcripts. Loss of bicoid mRNA was measured as a consequence. dcr-1 mutant eggs exhibit an impaired RNAi response to dsRNA and siRNA. Six-fold more bicoid mRNA was present in dcr-1 mutant eggs compared to wild-type eggs after either dsRNA or siRNA treatment. This result indicates that Dcr-1 acts downstream of siRNA production in the RNAi pathway. Dcr-1 plays an important though not absolute role in siRNA-dependent RNAi. Since Dcr-2 is also required downstream of siRNAs, these data suggest that Dcr-1 and Dcr-2 function might be partially redundant in some downstream activity (Lee, 2004).
Dcr-1 is essential to generate mature miRNAs. This role was demonstrated by analyzing miRNA levels in dcr-1 mutant eggs. No mature miRNAs belonging to the miR-2 group were detected in dcr-1 mutant eggs. Thus, Dcr-1 is critical for miRNA production whereas Dcr-2 is required primarily for siRNA production (Lee, 2004).
Although Dcr-1 and Dcr-2 preferentially produce different types of small RNAs, both are required for efficient siRNA-dependent mRNA degradation. Does this dual requirement extend to the miRNA pathway as well? To test this possibility, a genetic assay for miRNA-dependent gene silencing was used in dcr-1 and dcr-2 mutants. Several classes of motifs are present in the 3'UTR regions of the E(spl) and Bearded genes (Lai, 1998; Lai, 1997). The 3'UTR motifs are complementary to a variety of miRNAs, and they mediate posttranscriptional repression of gene expression (Lai, 2002). A series of reporter transgenes was constructed that mimics this posttranscriptional repression (Lai, 1998; Lai, 1997). The reporter genes contain a constitutive promoter from armadillo, lacZ coding sequence, and the 3'UTR from the Bearded or E(spl)m8 gene. When the reporter contains a wild-type Bearded 3'UTR, its expression in the developing eye disc is very weak. It is somewhat more strongly expressed in the eye disc posterior to the morphogenetic furrow and is equally weak in the anterior eye disc and antennal disc. When the reporter contains a Bearded 3'UTR with its three B motifs mutated, expression is ubiquitously strong in the eye and antennal discs, confirming that the B motifs mediate a silencing effect on gene expression (Lee, 2004).
Expression of a wild-type reporter gene was examined in clones of mutant dcr-2 cells that were generated in the developing eye disc. Clones expressed the reporter at levels indistinguishable from wild-type tissue, indicating that Dcr-2 is not required for this gene silencing mechanism. In contrast, expression of a wild-type reporter gene in clones of mutant dcr-1 cells was much stronger than in wild-type tissue. The derepressive effect of the dcr-1 mutation requires intact B motifs in the Bearded 3'UTR, since mutant clones did not affect expression of a reporter gene with mutated B motifs. These results argue that dcr-1 but not dcr-2 is necessary for posttranscriptional gene silencing that is mediated by a miRNA mechanism. This conclusion is also validated by other mutant phenotypes associated with each gene. Loss of dcr-1 has profound effects on Drosophila development within both somatic- and germ-lineages, whereas loss of dcr-2 appears to have little or no effect on development (Lee, 2004).
The dcr-1 mutant clones exhibited an interesting pattern of reporter expression. Clones in the antennal disc and eye disc, anterior to the morphogenetic furrow, exhibited little or no derepression of the reporter gene. Typically, only a few mutant cells in each clone had high levels of reporter gene expression. No overt cell differentiation occurs in this region of the eye disc. In contrast, almost all eye disc clones posterior to the furrow, where cell differentiation actively occurs, exhibited extensive derepression. Many, if not most, mutant cells in a clone exhibited this behavior. A boundary of reporter gene expression within a clone could be detected if the clone was bisected by the furrow. This boundary coincided with the morphogenetic furrow. Three interpretations seem possible. (1) Different sets of miRNAs repress the reporter in different regions of the eye disc, one set of which requires Dcr-1 and one set of which does not. However, all of these miRNAs would have to act through the 3'UTR binding sites, since a mutated reporter is constitutively derepressed. Thus, this interpretation is not favored. (2) Dcr-1 might not be essential in anterior disc cells because Dcr-2 or another factor substitutes if Dcr-1 is missing. (3) Anterior disc cells may contain miRNAs that were originally generated in dcr-1+ progenitor cells, and may therefore not require dcr-1. Passage of the morphogenetic furrow may trigger miRNA turnover, resulting in renewed dependence on dcr-1 posterior to the furrow (Lee, 2004).
The RNAi pathway can be divided into discrete biochemical steps: dsRNA processing, maintenance of siRNA 5' phosphate termini, siRNA loading into siRISC, and siRISC-based cleavage of target mRNA. It was confirmed that Dcr-2 but not Dcr-1 is required for dsRNA processing by incubating radiolabeled dsRNA substrate in lysates made from mutant embryos and monitoring siRNA formation. Labeled 21-23 nucleotide RNAs were readily detectable from reactions with wild-type and dcr-1 lysates, but were greatly reduced in reactions with dcr-2 lysate (Lee, 2004).
The genetic experiments suggested a role for Dcr-1 and Dcr-2 downstream of dsRNA processing. Therefore, lysates made from mutant embryos were assayed for steps downstream of siRNA production. siRNAs require 5' phosphate termini for proper association with RISC, and the 5' phosphates are maintained by a kinase that recognizes siRNAs (Nykanen, 2001). Both wild-type and dcr-2 mutant lysates efficiently converted synthetic siRNAs bearing 5' hydroxyl groups into 5'-phosphorylated forms, indicating that the mutant lysates have normal end-maintenance activity (Lee, 2004).
Nykanen (2001) reported that siRNAs are incorporated into a 350 kDa complex when incubated with embryo lysate. The complex is converted to active siRISC using ATP hydrolysis. Radiolabeled siRNA was incubated with wild-type or dcr-2 mutant embryo lysates in the presence of ATP, and then the products were size-fractionated by gel filtration chromatography. siRNA was predominantly associated with a 350 kDa complex in wild-type lysate. However, most of the siRNA in the dcr-2 mutant lysate fractionated as if unbound by proteins, indicating that Dcr-2 is essential for siRNA entry into functional complexes. Thus, dcr-2 lysates should be defective for siRNA-directed mRNA cleavage. SiRNA complementary to a radiolabeled target mRNA was coincubated with embryo lysate. Incubation in wild-type lysate produced a truncated mRNA whose length was consistent with it being the 5' cleavage product. Incubation in dcr-2 lysate generated 5- to 15-fold less cleavage product. This indicates that Dcr-2 is required for mRNA target cleavage by a siRNA, and is consistent with the in vivo observations (Lee, 2004).
To examine siRISC formation in dcr-1 mutant lysate, native gel electrophoresis was used. Gel filtration chromatography requires large lysate volumes, which could not be obtained from dcr-1 embryos. Pham (2004) has developed a gel electrophoretic method to characterize siRNA complexes. The R1 gel complex corresponds to Dcr-2 and R2D2 proteins bound to labeled siRNA. R2 complex appears to be an intermediate that links R1 to a third complex, R3. The R3 complex corresponds to a siRISC that is competent to cleave cognate mRNA (Pham, 2004). To address the role of Dcr-1 in complex formation, complexes were sought in a dcr-1 mutant lysate. R1 complex was detected, but its mobility was slightly shifted and more heterogeneous. No complex with comparable mobility to R2 was detected. The data indicate that proper formation of the R2 intermediate from the R1 precursor complex is dependent upon Dcr-1 (Lee, 2004).
Bacterial RNase III is an antiparallel dimer containing a deep cleft within the catalytic domain. At each end of the cleft lies a symmetric cluster of acidic residues that are conserved among RNase III enzymes. Some of the residues at each end coordinate a divalent metal ion (Mg2+) that is essential for the nucleophilic attack on the RNA phosphodiester bonds at each active site. The metal ion makes a bidentate interaction (inner-sphere and outer-sphere) with an invariant glutamate residue. In one model of catalysis, the nucleophile is used twice, thereby cleaving both strands. In another model, based on a crystal structure, each active site uses two separate clusters of residues to cleave the two phosphodiesters of the RNA helix. One cluster involves the metal ion, and the other cluster acts independent of the metal ion (Lee, 2004 and references therein).
Dicers contain two catalytic domains, which based on the structure of bacterial RNase III, might fold into a pseudodimer structure or might associate within a dimeric holoenzyme. In either scenario, the catalytic repeats contain many of the invariant acidic residues implicated in RNase III catalysis. Moreover, like bacterial RNase III, Dicers cleave dsRNA to produce fragments with 3' overhangs, and with 5'-phosphate and 3'-hydroxy termini in a reaction that requires a divalent metal ion. On this basis, the chemistry of phosphodiester hydrolysis is likely to be similar. Accordingly, certain invariant residues were mutated within Dcr-2 that ware predicted to specifically disrupt phosphodiester hydrolysis. E1371 and E1617 in the first and second RNase III repeats, respectively, are homologous to the E residue that extensively interacts with Mg2+ in the bacterial holoenzyme. An E->K mutant in E. coli RNase III fails to cleave dsRNA, but still binds the dsRNA substrate. To elucidate the functions of the homologous residues in Dcr-2, single and double E1371K and E1617K substitution mutants of dcr-2 were generated and the mutant genes were transformed into a Drosophila strain null for dcr-2. Their ability to silence white expression in a GMR-wIR background was tested. The single mutants gave barely detectable silencing activity, while the double mutant gave no detectable silencing. Thus, E1371 and E1617 are essential for Dcr-2 activity in vivo (Lee, 2004).
Two other conserved acidic residues in bacterial RNase III form an interdomain bridge near each metal binding site, but do not coordinate the metal. Interestingly, all known Dicers have acidic residues in homologous positions of the first domain repeat, but have nonacidic residues in the homologous positions of the second domain repeat. To test the functionality of the residues in the first domain, an E1210V or E1237A substitution was introduced into the dcr-2 gene. The same substitutions at the homologous positions of E. coli RNase III abolish activity. However, both dcr-2 point mutants fully rescued the dcr-2 null phenotype, indicating that E1210 and E1237 are not critical for Dcr-2 activity. These results are consistent with the notion that the nonacidic partner residues in the second repeat normally render these clusters nonfunctional (Lee, 2004).
These genetic and biochemical analyses support the idea that siRISC activity is dependent on Dcr-1 and Dcr-2. Pham (2004) has observed both Dcr-1 and Dcr-2 proteins in siRISC that is competent for target cleavage. At least three functions that Dicer could play in siRISC can be imagined. (1) Dicer may stably associate with siRISC after having passed an siRNA molecule to other RISC factors, but has no further role in siRISC activity. This is unlikely since siRNAs can UV-crosslink to Dcr-1 and Dcr-2 in assembled siRISC (Pham, 2004). (2) Dicer may use its dsRNA binding activity to retain double-stranded siRNA or a siRNA/mRNA duplex within siRISC. (3) The RNase III domain of Dicer may be responsible for RNA cleavage by siRISC. To test this latter possibility, the E1371K and E1617K variants of Dcr-2 were examined for siRISC activity (Lee, 2004).
Lysates were prepared from mutant embryos in which dcr-2E1371K, dcr-2E1617K, or dcr-2E1371K E1617K genes were expressed in place of the endogenous dcr-2 gene. Lysates were incubated with siRNA duplexes and a labeled mRNA substrate, and siRNA-directed cleavage of the substrate was monitored by 5' product formation. All three mutants exhibited normal mRNA cleavage activity in vitro. To demonstrate that the mutant proteins are nevertheless defective for RNase III activity, they were tested for dsRNA processing. Neither dcr-2E1371K nor dcr-2E1617K lysates were able to support dsRNA cleavage to form siRNAs. These data indicate that siRISC activity is unaffected when Dcr-2 RNase III activity is specifically impaired (Lee, 2004).
Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. This study demonstrates that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. The work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals (Wang, 2006).
RNA interference (RNAi) silences gene expression through small interfering RNAs (siRNAs) and microRNAs (miRNAs). In Drosophila, Dicer-2 (Dcr-2) produces siRNAs, whereas Dicer-1 (Dcr-1) recognizes precursors of miRNAs. The small RNAs are assembled with an Argonaute (Ago) protein into related effector complexes, such as RNA-induced silencing complex (RISC), to guide specific RNA silencing (Wang, 2006).
RNA silencing provides an antiviral mechanism in plants and animals. Plant viruses have evolved diverse strategies for evading the RNA silencing immunity, and expression of viral suppressors of RNAi (VSRs) is essential for infection and virulence. However, it is unknown whether antiviral silencing in plants is controlled by a specific small RNA pathway targeted by plant VSRs. Bacterial and fungal infections of Drosophila induce Toll and immune deficiency (Imd) pathways, leading to transcriptional induction of antimicrobial peptide effectors via NF-KappaB)like signaling processes. However, it has been unclear whether either pathway plays a role in Drosophila innate immunity against viruses. Previous work in cell culture has indicated that RNAi might mediate viral immunity in Drosophila. This study investigated whether RNAi indeed provides protection against virus infection in Drosophila embryos and adults (Wang, 2006).
Flock house virus (FHV) contains an RNA genome divided among two plus-strand molecules, RNAs 1 and 2. RNA2 (R2) encodes the single virion structural protein, whereas RNA1 (R1) encodes protein A, the viral RNAdependent RNA polymerase (RdRP), and B2, a VSR expressed after RNA1 replication from its own mRNA, RNA3. In the absence of R2, R1 replicates autonomously, accumulates to high levels, and produces abundant RNA3 in wild-type (WT) Drosophila embryos 30 hours after injection with R1 transcripts synthesized in vitro. No FHV RNAs accumulate in WT embryos injected with R1fs transcripts that contain a frameshift mutation in the RdRP open reading frame (ORF). FHV RNAs are also not readily detected in WT embryos injected with a second mutant of R1, R1DeltaB2, which does not express the VSR. However, abundant accumulation of R1DeltaB2 but not FR1fs occurs in mutant Drosophila embryos that carry a homozygous null mutation in ago-2 (ago-2414), which is essential for RNAi in Drosophila. These data indicated that viral RNA replication in Drosophila embryos triggers an RNAi-mediated virus clearance in an Ago-2dependent manner and that effective RNAi suppression by B2 is necessary to achieve normal accumulation of FHV RNAs (Wang, 2006).
In Drosophila, Ago-2 acts downstream of Dicer-2 (Dcr-2) to recruit siRNAs, the products of Dcr-2 activity, into the siRNA-dependent RISC (siRISC). Thus, a genetic requirement for ago-2 in FHV RNA clearance implicates Dcr-2 in the RNAi antiviral effector mechanism. To test this hypothesis, R1, R1fs, and R1DeltaB2 transcripts were injected into embryos carrying a homozygous dcr-2 null mutation, dcr-2L811fsX. Northern blot hybridizations showed that, although FHV RNAs remained undetectable in dcr-2L811fsX embryos injected with R1fs, viral RNA accumulation is rescued in the dcr-2L811fsX embryos injected with R1DeltaB2 transcripts. This result shows that Dcr-2 is required to initiate RNAi-mediated clearance of FHV RNAs in Drosophila embryos (Wang, 2006).
To investigate whether the RNAi pathway protects Drosophila from virus infection, adult flies of either WT or dcr-2L811fsX genotype were injected with purified FHV virions. The FHV isolate was of low virulence in WT flies, because about 50% of infected flies survived 15 days postinoculation (dpi) despite a detectable virus load. Inoculation with the same dose of FHV resulted in 60% mortality by 6 dpi and 95% by 15 dpi in dcr-2L811fsX flies. Mock inoculation with buffer had little effect on either WT or dcr-2L811fsX flies for as long as observations were made. Both Northern and Western blot analyses revealed that the virus accumulated more rapidly and to much greater levels in dcr-2L811fsX than WT flies. Thus, dcr-2 mutants exhibit enhanced disease susceptibility to FHV in comparison with WT flies, demonstrating that Dcr-2 is also required to mount an immune response that protects adult Drosophila against FHV infection (Wang, 2006).
R2D2 contains tandem double-stranded RNA (dsRNA)binding domains and forms a heterodimer with Dcr-2 in vivo that is required for siRNA loading into RISC. Flies homozygous for a loss-of-function mutation in r2d2 exhibit a phenotype of enhanced disease susceptibility to FHV infection similar to that of dcr-2L811fsX. Thus, R2D2 also participates in the innate immunity pathway that protects adult flies from FHV infection. Notably, although FHV accumulates to extremely high levels in both dcr-2 and r2d2 mutant flies, abundant viral siRNAs were detected only in r2d2 mutant flies, and viral siRNAs were below the level of detection in dcr-2L811fsX flies. Thus, FHV infection is detected by Dcr-2, leading to production of FHV siRNAs. However, R2D2 is not required for the production but is essential for the function of viral siRNAs, which is consistent with the genetic requirements for processing the artificially introduced dsRNA (Wang, 2006).
To investigate whether the RNAi pathway in Drosophila is specific against nodaviruses and not other classes of RNA viruses, the susceptibility of WT, dcr-2L811fsX, and r2d2 mutant flies to cricket paralysis virus (CrPV) was assessed. CrPV contains a nonsegmented plus-strand RNA genome but belongs to a group of picorna-like viruses. CrPV is substantially more virulent than FHV in Drosophila; injection of CrPV at much lower titers resulted in mortality of 70% of WT flies by 15 dpi. CrPV was also found to induce enhanced disease susceptibility in both dcr-2 and r2d2 mutant flies. About 60% of the infected mutant flies were dead by 6 dpi, and more than 95% were dead by 15 dpi. In addition, Northern blots indicated that the virus accumulated more rapidly and to greater levels in the mutant flies. Thus, both dcr-2 and r2d2 are required for protection of Drosophila against CrPV (Wang, 2006).
CrPV infection of cultured S2 cells induced antiviral silencing, illustrated by detection of CrPV-specific siRNAs. Antiviral silencing against FHV in S2 cells induced by FR1gfp is suppressed by CrPV superinfection, leading to derepression of green fluorescent protein (GFP). Two ORFs are encoded by the CrPV RNA genome. No suppression of antiviral silencing was observed in S2 cells cotransfected with a plasmid expressing either the entire downstream ORF of CrPV or the individual mature virion proteins processed from the polyprotein. In contrast, RNAi suppression was detected after cotransfection with a plasmid expressing either the entire upstream ORF of CrPV or its N-terminal 140 codons. However, the suppressor activity was not detected after a frameshift mutation was introduced into pA, thus identifying the N-terminal fragment of 140 amino acids of the CrPV nonstructural polyprotein as a VSR (Wang, 2006).
In Drosophila, Imd signaling is stimulated by Gram negative (Gram) bacterial infection, whereas Toll signaling is triggered by Gram positive (Gram+) bacterial infection. To determine whether loss of the RNAi pathway initiated by Dcr-2 has an impact on the Toll and Imd signaling processes, WT, dcr-2L811fsX, and r2d2 mutant flies were subjected to immune challenge by inoculation with Escherichia coli (Gram) or Micrococcus luteus (Gram+). Northern blot hybridizations detected substantial transcriptional induction of the antimicrobial peptide gene Diptericin A 6 hours postimmune challenge (hpi) with either E. coli or M. luteus, which declined at 24 hpi as described. Similar induction patterns for Diptericin A were observed in dcr-2L811fsX and r2d2 mutant flies inoculated with Gram+ and Gram bacteria. Furthermore, it was found that induction of either Attacin A or Drosomycin by Gram+ and Gram bacteria was also not altered in dcr-2L811fsX and r2d2 mutant flies as compared to WT flies. These results indicate that induction of antimicrobial peptide genes via Toll and Imd signaling pathways is not compromised in dcr-2L811fsX and r2d2 mutant flies (Wang, 2006).
Nodaviruses and the polio-like CrPV belong to two different superfamilies of animal RNA viruses. The same set of RNAi pathway genes is required for Drosophila defense against FHV and CrPV and both viruses encode a potent VSR. These results collectively show that RNAi pathway functions as a common viral immunity mechanism in Drosophila and that RNAi suppression represents a general counterdefensive strategy used by insect viruses. Furthermore, a genetic requirement for Dcr-2, R2D2, and Ago-2 in antiviral silencing establishes a molecular framework for the innate immunity against viruses in Drosophila. None of Dcr-2, R2D2, and Ago-2 plays a detectable role in either the production or function of miRNAs in Drosophila. Thus, this work identifies the dsRNA-siRNA pathway of RNAi as providing the innate immunity against virus infection in Drosophila and establishes that dsRNA produced during virus replication acts as the pathogen trigger whereas Dcr-2 and Ago-2 act as host sensor and effector of the immunity, respectively. These results support and extend the previous findings on antiviral silencing in C. elegans (Wang, 2006).
Although NF-KappaB-like signaling in the Toll and Imd pathways do not appear to play a role in the RNAi-directed viral immunity mechanism in Drosophila, the fly mutant defective in the Janus kinase (JAK) Hopscotch exhibit a modest increase in susceptibility to infection with Drosophila C virus, suggesting an antiviral role for JAKsignal transducer and activator of transcription (STAT) signaling. Nonetheless, it is believed that RNAi-based immunity is independent of JAK-STAT signaling, because virus infection is not known to induce the RNAi pathway in Drosophila and FHV induction of the JAK-STAT responsive gene vir-1 is unaltered in the dcr-2 and r2d2 mutants. Because the Toll and Imd pathways are highly conserved in vertebrates, the Drosophila model established for RNAi may also be useful for the analyses of the innate antiviral immunity in vertebrates (Wang, 2006).
P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).
The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).
Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).
The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).
The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).
The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).
The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).
P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).
The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).
Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).
The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).
Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).
What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).
Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).
RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).
An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).
The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).
The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).
With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).
Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).
Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are ~24-30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity. This study describes a second system that represses Drosophila TEs by using endogenous small interfering RNAs (siRNAs), which are 21 nucleotide, 3'-end-modified RNAs that are dependent on Dicer-2 and Argonaute-2. In contrast to piRNAs, the TE-siRNA system is active in somatic tissues, and particularly so in various immortalized cell lines. Analysis of the patterns and properties of TE-derived small RNAs reveals further distinctions between TE regions and genomic loci that are converted into piRNAs and siRNAs, respectively. Finally, functional tests show that many transposon transcripts accumulate to higher levels in cells and animal tissues that are deficient for Dicer-2 or Argonaute-2. It is concluded that Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs) (Chung, 2008).
Although the Drosophila RNAi pathway produces regulatory siRNAs in response to viral invasion, exogenous dsRNA, or IR transgenes, relatively little is historically known about the endogenous usage of Drosophila RNAi. This study describes a rich set of bona fide siRNAs that derive from transposable elements in Drosophila. These data add to a host of concurrent studies that recently elucidated multiple classes of siRNAs that derive from the host genome, not only from TEs, but also from 3' cis-natural antisense gene pairs, long IR transcripts, and two unique intronic and exonic clusters localized to the klarsicht and thickveins genes. Although these myriad siRNAs differ in origin, with some derived from bidirectional transcription and others from intramolecular dsRNA, they are united by their dependence on Dcr-2 and Ago2, their 3'-end modification, and, for at least some members of each class, an appreciable dependence on Loqs (Chung, 2008).
TE-siRNAs may be confidently distinguished from previously described TE-piRNAs on the basis of their characteristic sizes, genomic origins, tissue distribution, and origin from within a given TE. Both types of small-RNA pathways are demonstrably required to restrict TE transcript accumulation, and their separable roles correlate with their distinct tissue requirements. The germline is highly active in TE-piRNA production and uses piRNA components to restrict TE accumulation, whereas somatic tissues such as adult heads specifically produce TE-siRNAs and use RNAi components to restrict TE levels. Similar conclusions on TE-siRNA biogenesis and function have been reached in the concurrent studies of other groups. Curiously, whereas the mouse male germline depends strongly on piRNAs to restrict transposon activity, the mouse female germline appears to use both piRNAs and siRNAs to control TE activity. Therefore, there has been evolutionary flux in how these conserved small-RNA pathways are used to control TEs in animals (Chung, 2008).
Curiously, it was found that independently derived lines of cultured cells, namely S2 and Kc, exhibit pronounced siRNA responses to a subclass of LTR retrotransposons. This can be directly correlated with the fact of deregulation and genomic amplification of these particular TEs. It is possible that transposon deregulation was a direct consequence of the process of cell immortalization. However, one could speculate that their deregulation was a gradual consequence of divorcing these cells from piRNA control, which in the animal occurs mostly in the germline and is transmitted from generation to generation via maternal deposition of piRNA complexes into the embryo. In either case, the stronger TE-siRNA response in cultured cells may be viewed as an adaptive response to deregulated transposons, as proposed for the piRNA pathway (Chung, 2008).
A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).
This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).
The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).
Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. This study demonstrates that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals (Toledano, 2012).
Many stem cells lose the capacity for self-renewal when removed from their local microenvironment (or niche), indicating that the niche has a major role in controlling stem-cell fate. Changes to the local and systemic environments occur with age that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. The stem-cell niche in the Drosophila testis is located at the apical tip, where both germline stem cells (GSCs) and somatic cyst stem cells are in direct contact with hub cells. Hub cells express the self-renewal factor Upd, which activates the JAK-STAT signalling pathway to regulate the behaviour of adjacent stem cells. Ageing results in a progressive and significant decrease in the levels of upd in hub cells. However, constitutive expression of upd in hub cells was sufficient to block the age-related loss of GSCs, suggesting that mechanisms might be in place to regulate upd and maintain an active stem-cell niche (Toledano, 2012).
To identify potential regulators of upd, a collection of transgenic flies carrying green fluorescent protein (GFP)-tagged proteins was screened for expression in hub cells. The Drosophila homologue of Imp protein is expressed throughout the testis tip in young flies (Fabrizio, 2008); however, antibody staining revealed a decrease (~50%) in Imp expression in the hub cells of aged males. Imp is a member of a conserved family of RNA-binding proteins that regulate RNA stability, translation and localization (Yisraeli, 2005). Given the similarity in the ageing-related decline in Imp protein and upd mRNA in hub cells, it is proposed that Imp could be a new regulator of upd (Toledano, 2012).
To address whether Imp acts in hub cells to regulate upd, the bipartite GAL4-UAS system was used in combination with RNA-mediated interference (RNAi) to reduce Imp expression exclusively in hub cells. Fluorescence in situ hybridization (FISH) to detect upd mRNA was used in combination with immunofluorescence microscopy to determine whether the loss of Imp expression affects upd levels. The loss of Imp specifically in hub cells resulted in reduced expression of upd, as well as a significant reduction in GSCs and hub cell), when compared with controls. Consistent with a reduction in JAK-STAT signalling, decreased accumulation of STAT was observed when Imp levels were reduced by RNAi in hub cells (Toledano, 2012).
RNA-binding proteins characteristically target several RNAs; therefore, it was of interest to determine whether upd is a physiologically relevant target of Imp. Expression of upd together with an Imp RNAi construct was sufficient to completely rescue the defects caused by reduced Imp expression in hub cells, suggesting that Upd acts downstream of Imp to maintain GSCs and niche integrity. Importantly, the constitutive expression of upd alone in hub cells did not lead to an increase in GSCs in testes from 1-day-old males. These data suggest that Imp acts in hub cells to promote niche integrity and GSC maintenance, at least in part, by positively regulating upd (Toledano, 2012).
If Imp acts in hub cells in adult testes to regulate upd mRNA, it is speculated that the loss of Imp function during development might lead to a decrease in upd and a subsequent reduction in GSCs. Null mutations in Imp result in lethality at the pharate adult stage; therefore, testes from third instar larvae (L3) carrying Imp null alleles, Imp7 and Imp8, were examined. Deletion of the Imp locus was verified by PCR of genomic DNA. Combined immunofluorescence and FISH showed that although Fas3+ hub cells were easily detected, the expression of upd was significantly reduced: 24% of Imp7 mutants and 15% of Imp8 mutants had no detectable upd at this stage. In addition, the average number of GSCs and hub cells in testes from Imp mutants was significantly reduced when compared with control L3 testes. Notably, the re-expression of Imp in somatic niche cells was sufficient to rescue upd expression in Imp mutants to comparable levels to controls, and the reduction in the average number of GSCs and hub cells in Imp mutants was also reversed (Toledano, 2012).
Imp family members contain conserved KH domains that mediate direct binding to RNA targets. To determine whether Imp could associate directly with upd mRNA in vivo, testes were dissected from young flies expressing GFP-tagged Imp. Immunoprecipitation of Imp with anti-GFP antibodies, followed by quantitative reverse transcriptase PCR (qRT-PCR) analysis, showed a significant enrichment (~208-fold) of associated upd mRNA relative to control antibodies. Minimal enrichment for the ubiquitously expressed RNAs rp49 (also known as RpL32; ~4-fold) and GapDH (also known as Gapdh1; ~8 fold) or for the negative control med23 (~4-fold), was observed after Imp immunoprecipitation, indicating that the interaction between Imp and upd mRNA in hub cells is specific. Consistent with these observations, Imp protein and upd RNA co-localized in hub cells within perinuclear foci, probably ribonucleoprotein particles (Toledano, 2012).
An in vitro protein-RNA binding assay showed that Imp associates with the upd 3' untranslated region (UTR), specifically the first 250 base pairs (region 1), as no substantial binding to other portions of the upd 3'UTR was detected. Moreover, Imp did not bind the 5' untranslated or coding regions of upd or to the med23 3'UTR. Notably, a putative consensus binding sequence CAUH (in which H denotes A, U or C) for the mammalian IMP homologues (IGF2BP1- 3) occurs 22 times within the upd 3'UTR, including a cluster of four tandem repeats within the first 35 nucleotides of region 1. To test whether this motif mediates binding between Imp and upd, the first 33 nucleotides were removed to generate a sequence excluding the CAUH repeats, which resulted in a reduction in binding, compare domain 1 with domain 2. Point mutations in the third nucleotide of each motif (U = G) did not abolish the binding; however, point mutations in the consensus motif of MRPL9 RNA, a target of mammalian IGF2BPs, also did not abolish binding, suggesting that secondary structures probably mediate the association between IGFBP family members and their target RNAs. Altogether, the data identify the first 33 base pairs of the upd 3'UTR as a putative target sequence for Imp, and support observations that Imp associates specifically with upd in vivo (Toledano, 2012).
To gain further insight into the mechanism by which Imp regulates upd, a GFP reporter was constructed that contained the 3'UTR from either upd or med23. Transcript levels for gfp were fivefold higher in Drosophila Schneider (S2) cells that co-expressed Imp with the gfp-upd-3'UTR reporter than in cells that co-expressed Imp with the gfp-med23-3'UTR reporter. The significant increase in reporter mRNA levels indicates that it is likely that Imp regulates upd mRNA stability (Toledano, 2012).
RNA-binding proteins, including mammalian IGF2BP1, have been shown to counter microRNA (miRNA)-mediated targeting of mRNAs. However, no consensus miRNA seeds were located within the first 34 base pairs of domain 1 of the upd 3'UTR. It is speculated that if Imp binding blocks small RNA-mediated degradation of upd, polyadenylated, cleaved upd degradation intermediates would be detected in the testes of older males, when Imp expression in hub cells is reduced. Using a modified rapid amplification of complementary DNA ends (RACE) technique, a specific cleavage product was identifed starting at nucleotide 33 of the upd 3'UTR in the testes of 30-day-old flies, but not in RNA extracts from the testes of 1-day-old males. Importantly, the same degradation product of upd was also detected in the testes of young flies when Imp was specifically depleted from hub cells using RNAi-mediated knockdown. As a positive control, the esi-2-mediated cleavage product of mus308 was detected in testes from both 1- and 30-day-old flies (Toledano, 2012).
To test whether small RNAs might mediate upd cleavage, small RNA libraries generated from the testes of 1- and 30-day-old flies were cloned and deep-sequenced. Although no small RNAs with exact pairing to the upd degradation product were identified, two short interfering RNAs (siRNAs; termed siRNA1 and siRNA2) with high sequence complementarity to the predicted target site in the upd 3'UTR were present in the testis library generated from 30-day-old males. Using qRT- PCR for mature small RNAs, it was found that the siRNA2 levels in the testes, relative to the levels of the control small RNAs bantam and mir-184, were similar in young and old males (deep sequencing analysis demonstrated that expression of these two control miRNAs did not change with age). The source of siRNA2 is the gypsy5 transposon, which is inserted at several loci throughout the fly genome and is conserved in numerous Drosophila species (Toledano, 2012).
To gain further insight into the mechanism by which Imp and siRNA2 regulate upd, the levels of the upd GFP reporter (gfp-upd-3'UTR) in the presence or absence of Imp and siRNA2 was investigated in S2 cells. To generate a reporter that should not be susceptible to siRNA-mediated degradation, the cleavage site in the upd 3'UTR that was identified by RACE (32AUU = CGG; gfp-upd-3'UTRmut) was mutated. Cells were transfected with either of the GFP reporter constructs, with or without haemagglutinin-tagged Imp (Imp- HA), and subsequently transfected with siRNA2; gfp expression was quantified by qRT- PCR (Toledano, 2012).
The co-expression of siRNA2 and the gfp-upd-3'UTR reporter resulted in a significant decrease in gfp transcript levels. Conversely, the co-expression of Imp blocked siRNA2-mediated reduction of gfp mRNA such that gfp levels were higher than in control cells. Furthermore, mutation of the putative cleavage site rendered the upd 3'UTR resistant to siRNA2-mediated degradation. These data, in combination with the in vitro binding data, suggest that Imp binds to and protects the upd 3'UTR from endogenous and exogenous siRNA2 in S2 cells. Thus, endo-siRNA2 is a bona fide candidate that could direct upd degradation when Imp is absent or its levels are reduced, although targeting by other small RNAs cannot be excluded (Toledano, 2012).
In Drosophila, Argonaute-1 (AGO1) is the principle acceptor of miRNAs and primarily regulates targets in a cleavage-independent mode, whereas AGO2 is preferentially loaded with siRNAs and typically regulates targets by mRNA cleavage. AGO2 expression was detected throughout the tip of the testis, as verified by immunostaining of testes from transgenic flies expressing 3×Flag-HA-tagged AGO2. To test whether AGO2 binds to upd mRNA in vivo, thereby potentially regulating upd levels directly, testes were dissected from aged (30-day-old) 3×Flag- HA- AGO2 males. Immunoprecipitation of AGO2, followed by qRT- PCR, showed significant enrichment (~102-fold) of upd mRNA bound to AGO2. Negligible binding of a negative control, rp49, to AGO2 was detected, suggesting specific association of AGO2 with upd mRNA in vivo and supporting a previous findings that upd is probably targeted by the siRNA pathway (Toledano, 2012).
To test whether Imp can impede the binding of AGO2 to the upd 3'UTR, S2 cells stably expressing Flag-tagged AGO2 were transfected with the gfp-upd-3'UTR reporter. Consistent with our previous observations, transcript levels of gfp-upd-3'UTR increased ~18-fold when Imp was co-expressed. Despite increases in the overall levels of gfp mRNA, the presence of Imp markedly reduced the association of AGO2 with the upd 3'UTR, indicating that Imp antagonizes the ability of AGO2 to bind the upd 3'UTR (Toledano, 2012).
Similar to the AGO family, Drosophila encodes two Dicer proteins that seem to have distinct roles in small RNA biogenesis. Dicer-1 (Dcr-1) is essential for the generation of miRNAs, and Dcr-2 is required for siRNA production from exogenous and endogenous sources. If siRNAs were involved in upd degradation in older males, it would be predicted that the loss of Dcr-2 would suppress the ageing-related decline in upd and GSCs. Consistent with a role for Dcr-2 in the generation of siRNAs, siRNA2 levels were significantly reduced in Dcr-2 homozygous mutants relative to heterozygous controls. Testes from 30- and 45-day-old Dcr-2 mutant flies showed increased levels of upd by qRT- PCR when compared with controls. Whereas a ~90% reduction of upd is observed in the testes from aged Dcr-2 heterozygous controls, only a ~45% reduction in upd was observed in testes from age-matched, Dcr-2 homozygous mutants, indicating that upd levels are higher when Dcr-2 function is compromised. Furthermore, the testes from aged Dcr-2 mutants contained more GSCs, on average, when compared with controls. Conversely, the forced expression of Dcr-2 in hub cells resulted in a reduction in the average number of GSCs and led to a significant reduction in upd levels, as detected using qRT- PCR and combined immunofluorescence and FISH, which seemed to be specific, as no significant change in Imp transcript levels was observed. Expression of Imp in combination with Dcr-2 resulted in a significant increase in upd levels. These observations indicate that Imp can counter the decrease in upd levels resulting from forced Dcr-2 expression, providing further evidence that Imp protects upd from targeted degradation by the siRNA pathway (Toledano, 2012).
The data suggest that Imp has a role in stabilizing upd in hub cells; therefore, the ageing-related decline in Imp would be a major contributing factor to the decrease in upd mRNA in the hub cells of aged males. To investigate the mechanism that leads to the decline in Imp expression with age, the Imp 3'UTR was examined for potential instability elements. Within the first 160 base pairs there is a canonical seed sequence for the heterochronic miRNA let-7. Expression of a reporter gene under the control of the let-7 promoter showed that let-7 expression increases in hub cells of ageing male, which was confirmed by let-7 FISH of testes from aged males. In addition, mature let-7 miRNA was enriched twofold in the testes from 30-day-old flies, relative to 1-day-old males. Therefore, an age-related increase in let-7 is one mechanism by which Imp expression could be regulated in an ageing-dependent manner in testes from older males (Toledano, 2012).
Consistent with these observations, the forced expression of let-7 specifically in hub cells led to a decrease in Imp. In addition, let-7 expression in S2 cells reduced the levels of a heterologous gfp-Imp-3'UTRWT reporter. S2 cells were transfected with a let-7 mimic or with negative control miRNA, and gfp expression was quantified by qRT- PCR. There was a 70% reduction in gfp-Imp-3'UTRWT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTRmut reporter with mutations in the canonical seed for let-7 (at nucleotide 137) was unaffected by let-7 expression, indicating that mutation of the let-7 seed rendered the RNA resistant to degradation. These data confirm that let-7 can destabilize Imp through sequences in the 3'UTR. However, further increasing the levels of let-7 resulted in a decrease in gfp expression from the mutated 3'UTR, indicating that other, putative let-7 seeds in the Imp 3'UTR can be targeted by let-7 (Toledano, 2012)
If the age-related decrease in Imp contributes to a decline in upd and subsequent loss of GSCs, it is proposed that re-expression of Imp in hub cells would rescue the ageing-related decrease in upd. Therefore, flies in which Imp was constitutively expressed in hub cells were aged, and upd levels were quantified by qRT- PCR. The expression of an Imp construct containing a truncated 3'UTR (Imp-KH- HA) lacking let-7 target sequences specifically in hub cells was sufficient to suppress the ageing-related decline in upd, with concomitant maintenance of GSCs, similar to what was observed by re-expressing upd in the hub cells of aged males. Maintenance of Imp-KH- HA expression in aged males was verified by staining with an anti-HA antibody. Conversely, the expression of an Imp construct that is susceptible to degradation by let-7 (ImpT21) did not lead to an accumulation of Imp in the testes of 30- and 50-day-old flies, as levels were similar to the levels of endogenous Imp at later time points. Consequently, the expression of this construct was not sufficient to block the ageing-related decline in GSCs. These data indicate that let-7-mediated regulation of Imp contributes to the decline in Imp protein in older flies, and supports a model in which an ageing-related decline in Imp, mediated by let-7, exposes upd to degradation by siRNAs. Thus, both the miRNA and siRNA pathways act upstream to regulate the ageing of the testis stem-cell niche by generating let-7 and siRNA2, which target Imp and upd, respectively (Toledano, 2012).
Drosophila has proven to be a valuable model system for investigating ageing-related changes in stem-cell behaviour. Cell autonomous and extrinsic changes contribute to altered stem-cell activity; thus, determining the mechanisms underlying the ageing-related decline of self-renewal factors, such as the cytokine-like factor Upd, may provide insight into strategies to maintain optimal niche function (Toledano, 2012).
The data indicate that Imp can regulate gene expression by promoting the stability of selected RNA targets by countering inhibitory small RNAs. Therefore, rescue of the aged niche by Imp expression may be a consequence of effects on Imp targets, in addition to upd, in somatic niche cells. Furthermore, as Imp is expressed in germ cells, it could also act in an autonomous manner to regulate the maintenance of GSCs. The canonical let-7 seed in the Imp 3'UTR is conserved in closely related species, and reports have predicted that the let-7 family of miRNAs target mammalian Imp homologues (IGF2BP1- 3). Given the broad role of the let-7 family in ageing, stem cells, cancer and metabolism, the regulation of Imp by let-7 may be an important, conserved mechanism in numerous physiological processes (Toledano, 2012).
Non-coding RNAs can ensure biological robustness and provide a buffer against relatively small fluctuations in a system. However, after a considerable change, a molecular switch is flipped, which allows a biological event to proceed unimpeded. In the current model, Imp preserves niche function in young flies until a time at which miRNAs and siRNAs act together to trigger an 'ageing' switch that leads to a definitive decline in upd and, ultimately, in stem-cell maintenance. Therefore, targeting signalling pathways at several levels using RNA-based mechanisms will probably prove to be a prevalent theme to ensure robustness in complex biological systems (Toledano, 2012).
Search PubMed for articles about Drosophila Dicer-2
Abrahante, J. E., Daul, A. L., Li, M., Volk, M. L., Tennessen, J. M., Miller, E. A. and Rougvie, A. E. (2003). The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs. Dev. Cell 4: 625-637. 12737799
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366. 11201747
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113: 25-36. 12679032
Caudy, A. A., Myers, M., Hannon, G. J. and Hammond, S. M. (2002). Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16: 2491-2496. 12368260
Caudy, A. A., et al. (2003). A micrococcal nuclease homologue in RNAi effector complexes. Nature 425(6956): 411-414. 14508492
Chung, W. J., Okamura, K., Martin, R. and Lai, E. C. (2008). Endogenous RNA interference provides a somatic defense against Drosophila transposons. Curr. Biol. 18(11): 795-802. PubMed Citation: 18501606
Czech, B., et al. (2008). An endogenous small interfering RNA pathway in Drosophila. Nature 453(7196): 798-802. PubMed Citation: 18463631
Djikeng, A., Shi, H., Tschudi, C., Shen, S. and Ullu, E. (2003). An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei. RNA 9: 802-808. 12810914
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date revised: 28 December 2011
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