Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2 (Forstemann, 2007).
Animal miRNAs are produced by the sequential action of two distinct RNase III endonucleases. Drosha converts primary miRNAs, most of which are full-length RNA polymerase II transcripts, into pre-miRNAs, 70 nt RNAs that fold into a stem-loop or hairpin structure. Dicer then excises the mature miRNA, bound to its miRNA* strand, from the pre-miRNA. In Drosophila, distinct Dicer enzymes produce siRNA and miRNA. Dicer-1 (Dcr-1) acts with a double-stranded RNA (dsRNA)-binding protein partner, Loquacious (Loqs), to convert pre-miRNA to a miRNA/miRNA* duplex, whereas Dicer-2 (Dcr-2) produces siRNA duplexes by cleaving long dsRNA. Dcr-2 also acts with its dsRNA-binding partner protein, R2D2, to load an siRNA duplex into Ago2, a function that is separable from its role in siRNA production (Forstemann, 2007).
Both siRNAs and miRNAs act as components of RNA-induced silencing complexes (RISCs); the core protein component of all RISCs is a member of the Argonaute family of small RNA-guided RNA-binding proteins. The Drosophila genome encodes five Argonaute proteins, which form two subclades. The Ago subclade comprises Ago1 and Ago2, which have been reported to bind miRNAs and siRNAs, respectively. Piwi, Aub, and Ago3 form the Piwi subclade of Argonaute proteins and bind repeat-associated siRNAs (rasiRNAs; also called piRNAs), which direct silencing of selfish genetic elements such as transposons (Forstemann, 2007).
In lysates from Drosophila embryos, in cultured Drosophila S2 cells, and in adult flies, miRNA can be loaded into both Ago1 and Ago2. The data suggest that sorting miRNAs into Ago1- and Ago2-RISC generates silencing complexes with distinct functional capacities: Ago1-RISC represses expression of targets with which its guide miRNA matches only partially, whereas Ago2 silences fully matched target RNAs. These differences result, in part, from the surprisingly different catalytic efficiencies of Ago1 and Ago2: only Ago2 catalyzes robust, multiple-turnover target cleavage (Forstemann, 2007).
In mammals, only Ago2 retains the ability to catalyze guide RNA-directed endonucleolytic cleavage of RNA; the three other mammalian Argonaute proteins, Ago1, Ago3, and Ago4, lack a functional active site that is presumed to have been present in the evolutionarily ancestral Argonaute protein. Why then has Drosophila Ago1 retained any endonuclease activity at all, if it is so inefficient at target cleavage that it cannot measurably contribute to small RNA-directed RNAi? One potential explanation is that the primary role of the Ago1 endonuclease activity is to facilitate loading of Ago1-RISC. That is, the predominant substrate for the Ago1 endonuclease is not target RNA but, rather, miRNA* strands and perhaps the occasional siRNA passenger strand. Because miRNA* strand cleavage would occur only in cis and only once per loaded Ago1-RISC, efficient, multiple-turnover cleavage of target RNA would not be required (Forstemann, 2007).
These data reveal an important biochemical difference between Ago2 and Ago1, but they do not explain the molecular basis for the inefficiency of Ago1-directed cleavage of target RNA. Two explanations can be envisioned for the more than 40-fold lower kcat of Ago1 compared to Ago2. First, the active site of Ago1 might be less well suited to catalyzing phosphodiester bond cleavage. Alternatively, Ago1 might be slow to assume a catalytically active conformation. In this second model, the rate of a conformational rearrangement would limit the speed of target RNA cleavage by Ago1 (Forstemann, 2007).
The genome of Drosophila contains no mRNA with complete complementarity to miR-277. Why then do flies load miR-277 into Ago2-RISC? Perhaps there are as yet unknown iral RNAs targeted by Ago2-loaded miR-277. Such an innate immune response function has previously been proposed for miRNAs in mammals. Regardless of the biological purpose for loading miR-277 into Ago2, miR-277 provides an important in vivo test of the controversial proposal that the production of small RNA duplexes by Dicer is uncoupled from the loading of Argonaute proteins. That Dcr-2 and R2D2 act in vivo to load Ago2 with miR-277, a miRNA produced by Dcr-1 and Loqs, confirms previous in vitro data suggesting that both ends of a small RNA duplex are available for examination by the Ago2 loading machinery. The results suggest that the miR-277/miR-277* duplex dissociates from Dcr-1 after the dicing of pre-miR-277 and is then bound by the Dcr-2/R2D2 heterodimer, which loads it into Ago2 (Forstemann, 2007).
It was reasoned that Ago1 loading is also uncoupled from dicing. In all animals, some miRNAs are found on the 5' and others on the 3' arm of their pre-miRNA stem loops. In contrast, the geometry of Dcr-1 with respect to the two arms of the pre-miRNA stem is essentially the same for all miRNAs: Dcr-1 always makes staggered cuts that separate the pre-miRNA loop from the miRNA/miRNA* duplex. If Dcr-1 were to load miRNAs directly into Ago1, without first releasing the miRNA/miRNA* duplex, it would be expected that all miRNAs would reside on the same arm of the pre-miRNA stem. The simplest explanation, and one most consistent with the partitioning of miR-277 into both Ago1- and Ago2-RISCs, is that miRNA/miRNA* duplexes are released from Dicer immediately after their production, then rebound by the Ago1- and Ago2-loading machineries. Such a model allows both the terminal thermodynamics of the miRNA/miRNA* duplex to determine the mature miRNA strand (rather than its position within the pre-miRNA) and the pattern of mismatches within the duplex to determine how the miRNA partitions between Ago1 and Ago2 (Forstemann, 2007).
In mammals, siRNAs produce off-target effects largely by acting like miRNAs. In flies, siRNAs loaded into Ago2 are believed to defend against viral infection. Virus-derived siRNAs might therefore trigger widespread, off-target silencing of host genes as flies mount an antiviral RNAi response. The partitioning of siRNAs into Ago2-RISC appears to circumvent this problem, because silencing by Drosophila Ago2 requires greater complementarity between the siRNA and its target than silencing by Ago1. It is tempting to speculate that a similar functional specialization among Argonaute proteins has gone undetected in mammals (Forstemann, 2007).
In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates the miRNA/miRNA* and siRNA duplexes from which Argonaute proteins are loaded. The Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA* duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures (Tomari, 2007).
In Drosophila the structure of a small-RNA duplex determines its partitioning between Ago1- and Ago2-RISC. These data suggest a simple model for this partitioning, with a central unpaired region serving as both an antideterminant for the Ago2-loading pathway and a preferred binding substrate for the Ago1 pathway. Supporting this view, miRNAs that contain central mismatches, such as let-7 and bantam, assemble primarily into Ago1-RISC. miR-277, whose central region is base paired, partitions between Ago1 and Ago2 in vivo (Tomari, 2007).
A model for small silencing RNA sorting in Drosophila. Dcr-2/R2D2 bind well to highly paired small-RNA duplexes but poorly to duplexes bearing central mismatches; such duplexes are therefore disfavored for loading into Ago2. Ago1 favors small RNAs with central mismatches, but no Ago1-loading proteins have yet been identified. Ago1- and Ago2-loading compete each other, increasing the selectivity of small-RNA sorting. The partitioning of a small-RNA duplex between the Ago1 and Ago2 pathways reflects its structure. A typical miRNA/miRNA* duplex, such as let-7 or bantam, loads mainly Ago1, whereas a standard siRNA duplex loads mostly Ago2. Some miRNA/miRNA* duplexes containing extensively paired central regions, such as miR-277/miR-277*, partition between Ago1 and Ago2. Sorting of small-RNA duplexes into Ago1 and Ago2 produces pre-RISC, in which the duplex is bound to the Argonaute protein. Subsequently, mature RISC, which contains only the siRNA guide or miRNA strand of the original duplex, is formed. The separation of the miRNA and miRNA* or the siRNA guide and passenger strands also reflects the structure of the small-RNA duplex. For Ago1, it is hypothesized that mismatches between the miRNA and the miRNA* or siRNA guide and passenger strands in the seed sequence are required for the efficient conversion of pre-RISC to mature RISC. For Ago2, such seed sequence mismatches are not needed because Ago2 can efficiently cleave the passenger or miRNA* strand, liberating the guide or miRNA from the duplex (Tomari, 2007).
Both the Ago2- and Ago1-loading pathways are selective. For Ago2, the affinity of the Dcr-2/R2D2 heterodimer for a small-RNA duplex provides the primary source of small-RNA selectivity. In the absence of either the Ago2-loading machinery or Ago2 itself, Ago1 is nonetheless preferentially loaded with a miRNA/miRNA* duplex; an siRNA duplex still loads poorly into Ago1. Thus, the Ago1-loading pathway is also inherently selective and not a default pathway that assembles small RNAs rejected by the Ago 2 pathway. It is not yet know if this selectivity is a direct property of Ago1, of an Ago1-loading machinery that remains to be identified, or both (Tomari, 2007).
Previous bioinformatic analyses noted that a central region of thermodynamic instability was a common feature of miRNA/miRNA* duplexes. The current data ascribe a function in flies to this common miRNA/miRNA* structural feature: directing the miRNA into Ago1 and away from Ago2. Mammalian miRNA/miRNA* duplexes also typically contain a central unpaired region, but it is not yet known if they are preferentially loaded into one of the four mammalian Ago-subclade Argonaute proteins (Tomari, 2007).
What is the biological significance in flies of sorting miRNAs into Ago1 and siRNAs into Ago2? One idea is that Ago1 and Ago2 are functionally distinct, with only Ago2 silencing targets that possess extensive complementarity to the small-RNA guide and only Ago1 directing repression of targets that contain multiple but only partially complementary miRNA-binding sites. Sorting small RNAs between Ago1 and Ago2 may also prevent miRNAs from saturating the Ago2 machinery, which might compromise Ago2-mediated antiviral defense. Conversely, excluding from Ago1 siRNAs produced in response to viral infection may minimize competition between such antiviral siRNAs and endogenous miRNAs, protecting flies from misregulation of gene expression during a viral infection. Restricting a robust RNAi (i.e., target cleavage) response to siRNAs loaded into Ago2 may also minimize undesirable, miRNA-like regulation of cellular genes by virally derived siRNAs. Thus, small-RNA sorting ensures that miRNAs are largely restricted to Ago1, whose relaxed requirement for complementarity between a miRNA and a regulated mRNA target allows each miRNA to control many different mRNAs, and that siRNAs are restricted to Ago2, whose silencing activity requires more extensive complementarity between the target and the siRNA guide. Nonetheless, a final question remains unanswered: why do some iconoclastic miRNA/miRNA* duplexes contain features that favor their loading into Ago2 (Tomari, 2007)?
Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways. In the RNA interference (RNAi) pathway, small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons. This study reports that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs and mouse and rat piRNAs. Plant small-RNA methylation is catalyzed by the protein HEN1. Drosophila melanogaster Hen1 (DmHen1), the Drosophila homolog of HEN1, termed Pimet (piRNA methyltransferase) by Saito (2007) in a parallel study, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs, which derive from double-stranded precursors, and piRNAs which do not. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after the Argonaute-bound siRNA duplex is converted to single-stranded RNA (Horwich, 2007; Saito, 2007).
In flies, both piRNAs (also known as repeat-associated siRNAs, rasiRNAs) and siRNAs, but not miRNAs, are modified at their 3' termini. The terminal nucleotide of Drosophila 0-2 hr embryo and mouse and bull testicular piRNAs was selectively labelled. The resulting 32P-radiolabeled nucleoside 2' or 3'-monophosphates were resolved by 2D thin-layer chromatography (2D TLC) with a solvent system that can resolve nucleoside 2' monophosphates, nucleoside 3' monophosphates, and 2'-O-methyl nucleoside 3' monophosphates. Modified nucleoside monophosphates derived from the 3' termini of piRNAs were identified by comparison to modified and unmodified nucleoside 2' and 3' monophosphate standards. The terminal nucleotide of the piRNAs of all three animals comigrate with 2'-O-methyl nucleoside 3' monophosphate standards but not with any unmodified nucleoside monophosphate standard. Because mouse piRNAs were previously shown to contain 2'-O-methyl modified 3' termini by both mass spectrometry and a 2D TLC system, it is concluded that Drosophila and bull piRNAs also contain a 2'-O-methyl group at their 3' termini (Horwich, 2007).
In Arabidopsis, the RNA methyltransferase, HEN1, modifies the terminal 2' hydroxyl group of small silencing RNAs. In Drosophila, predicted gene CG12367, whose 1559 nucleotide mRNA encodes a 391 amino acid protein with a 220 amino acid evolutionarily conserved methyltransferase domain, most closely resembles Arabidopsis HEN1. For simplicity, this gene has been called Drosophila melanogaster (Dm) hen1. When homozygous, a piggyBac transposon insertion (PBac{WH}CG12367[f00810]) within the first intron of the fly hen1 gene reduces the accumulation of hen1 mRNA by 1000-fold in testes and by more than 40,000-fold in ovaries and can therefore be considered a null mutation, which is referred to as hen1f00810 (Horwich, 2007).
The 3' termini of two types of highly abundant piRNAs were examined in the germline of flies heterozygous or homozygous for hen1f00810. In testes, the Suppressor of Stellate [Su(Ste)] locus produces 24-27 nucleotide rasiRNAs, a subclass of piRNAs that directs silencing of the selfish genetic element Stellate. Su(Ste) rasiRNAs, like other Drosophila piRNAs, are modified at their 3' termini and therefore do not react with NaIO4. In contrast, Su(Ste) rasiRNAs from hen1f00810/hen1f00810 mutant testes reacted with NaIO4 and could therefore be β-eliminated to remove the last nucleotide of the RNA, thereby increasing their gel mobility and indicating that in the absence of DmHen1 protein, they are not modified. Similarly, rasiRNAs that guide silencing of roo, the most abundant retrotransposon in Drosophila melanogaster, were not modified in hen1f00810 homozygous ovaries. The Su(Ste) and roo rasiRNAs were also shorter in the hen1f00810 homozygotes. In contrast, the length and amount of miR-8, which is expressed in both the male and female germline, was unaltered in hen1f00810 homozygotes. For both Su(Ste) and roo, rasiRNAs were on average shorter and less modified even in hen1f00810 heterozygotes, compared to the wild-type, suggesting that the abundance of DmHen1 protein limits the stability or production of piRNAs in flies (Horwich, 2007).
Modification of the termini of Drosophila piRNAs plays a role in their function: mRNA expression from HeT-A, the element whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline, quadrupled in hen1f00810 heterozygotes and was increased by more than 11-fold in homozygotes, relative to wild-type tissue. It is concluded that Hen1 protein is required for piRNA-directed silencing in the Drosophila germline (Horwich, 2007).
To test whether DmHen1 is required for modification of the 3' termini of siRNAs, Hen1 was depleted by RNAi in cultured Drosophila S2 cells. The cells were transfected with long double-stranded RNA (dsRNA) targeting hen1 on day 1 and day 5, then cotransfected with both GFP dsRNA and hen1 dsRNA on day 8. Total RNA was harvested on day 9, probed for modification with NaIO4/β-elimination, and analyzed by Northern hybridization with a 5' 32P-radiolabeled DNA probe complementary to the most abundant GFP-derived siRNA. DsRNAs targeting two different regions of the fly hen1 mRNA both reduced the amount of GFP siRNA modified at its 3' terminus, whereas all the GFP siRNA remained modified when a control dsRNA was used (Horwich, 2007).
Surprisingly, RNAi-mediated depletion of Ago2, but not Ago1, prevented the GFP siRNA from being modified. This result suggests that Ago2, but not Ago1, plays a role in the modification of siRNAs by DmHen1. To test this idea, the modification status of the 3' terminus of miR-277, which partitions into both Ago1 and Ago2 complexes in vivo, was examined. Drosophila miRNAs associate predominantly or exclusively with Ago1 and have unmodified 3' termini. In contrast, approximately half the miR-277 in cultured S2 cells failed to react with NaIO4, suggesting that approximately half of miR-277 is modified at its 3' terminus. The fraction of miR-277 that was modified was reduced when two different dsRNAs were used to deplete DmHen1 by RNAi. When the cells were treated with dsRNA targeting ago1, all detectable miR-277 was modified, whereas all miR-277 became unmodified when dsRNA targeting ago2 was used. In contrast, bantam, a miRNA that associates nearly exclusively with Ago1, was unmodified under all conditions (Horwich, 2007).
siRNA modification can be recapitulated in lysates of embryos, ovaries, or cultured S2 cells. Modification of siRNA in vitro was inhibited by S-adenosyl homocysteine, but not by S-adenosyl methionine, consistent with DmHen1 transferring a methyl group from S-adenosyl methionine to the terminal 2' hydroxyl group of the RNA, thereby generating S-adenosyl homocysteine as a product (Horwich, 2007).
Data from cultured S2 cells suggested that DmHen1 modifies that portion of miR-277 that enters the Ago2-RISC-assembly pathway, but not the population of miR-277 that assembles into Ago1-RISC. To further test the idea that small-RNA modification requires both Hen1 and the Ago2-RISC-assembly pathway, cytoplasmic lysates were prepared from dsRNA-treated cultured S2 cells. Lysate from control-treated cells modified the 3' terminus of a 5' 32P-radiolabeled synthetic siRNA duplex but not lysate from hen1-depleted cells. The addition of either of two different preparations of purified, recombinant DmHen1, expressed in E. coli as a ~74 kDa glutathione S-transferase fusion protein (GST-DmHen1), restored the ability of the lysates to modify the siRNA, indicating that loss of DmHen1 caused the loss of siRNA modification. Moreover, lysates depleted for Ago2, but not Ago1, could not modify the 32P-siRNA in vitro. These in vitro data, together with S2-cell experiments, suggest that modification of the 3' terminus of siRNAs and miRNAs is coupled to assembly into Ago2-RISC (Horwich, 2007).
Dcr-2 and R2D2 act to load double-stranded siRNAs into Ago2. Lysates were prepared from ovaries homozygous mutant for hen1, dcr-2, r2d2, and ago2 by using alleles that were unable to produce the corresponding protein. A 5' 32P-radiolabeled siRNA duplex was incubated in each lysate to assemble RISC. At each time point, whether the siRNA was 3' terminally modified was determined by assessing its reactivity with NaIO4. No modified siRNA accumulated when the duplex was incubated in hen1f00810, dcr-2L811fsX, r2d21, or ago2414 mutant lysate. Adding 250 nM purified, recombinant GST-DmHen1 restored siRNA modification to the hen1f00810 but not the ago2414 lysate. It is concluded that the defect in ago2414 reflects a requirement for Ago2 in small-RNA modification by DmHen1, rather than an indirect effect such as destabilization of DmHen1 in the absence of Ago2. GST-DmHen1 similarly rescued lysate from hen1(RNAi) but not ago2(RNAi)-treated S2 cells. Together, the results of experiments using cultured S2 cells -- a somatic-cell line -- and ovaries, which comprise mainly germline tissue, suggest that a functional Ago2-RISC-assembly pathway is required for siRNA modification in Drosophila (Horwich, 2007).
To test at which step in the Ago2-RISC-assembly pathway siRNAs become modified, it was determined whether siRNAs are 2'-O-methylated by DmHen1 as single strands or as duplexes. In vitro, assembly of siRNAs into Ago2-RISC follows an ordered pathway in which the siRNA duplex first binds the Dicer-2/R2D2 heterodimer to form the RISC-loading complex (RLC). The RLC determines which of the two siRNA strands will become the guide for Ago2 and which will be destroyed (the passenger strand). The siRNA is then loaded into Ago2 as a duplex. In this pre-RISC complex, the passenger strand occupies the same position as future target RNAs. Cleavage of the passenger strand by the Ago2 endonuclease domain converts pre-RISC to mature RISC. No single-stranded guide or passenger RNA is produced prior to this maturation step. Thus, all single-stranded siRNA produced in vitro or in vivo corresponds to mature RISC (Horwich, 2007).
Ago2-RISC was assembled in vitro by using an siRNA designed to load only one of its two strands into Ago2. Then the reaction was sampled over time, isolating the 5' 32P-radiolabeled siRNA under conditions previously demonstrated to preserve its structure, and single- from double-stranded siRNA was separated by native gel electrophoresis. The RNAs were then isolated from the gel and tested for reactivity with NaIO4 to determine the presence of modification at their 3' termini. At each time, total siRNA was analyzed in parallel. 3' terminal modification increased over the course of RISC assembly and, at all times, was restricted to single-stranded siRNA: Within the limits of detection, all double-stranded siRNA was unmodified, even after 3 hr. It is concluded that siRNA modification is coupled to RISC assembly and occurs only after the conversion of pre-RISC to mature RISC (Horwich, 2007).
Whereas Arabidopsis HEN1 contains an N-terminal double-stranded RNA-binding motif, DmHen1 does not. To test whether DmHen1 modifies double-stranded small RNAs, purified, recombinant GST-DmHen1 was incubated with either single-stranded or double-stranded siRNAs. Modification, evidenced by loss of reactivity with NaIO4, was detected only for the single-stranded RNA, suggesting that DmHen1 modifies single-stranded substrates, but not siRNAs or blunt RNA duplexes. A preference for single-stranded RNA would explain how DmHen1 could act on both siRNAs, which are born double stranded, and piRNAs, which are not. It is noted that the purified, recombinant GST-DmHen1 protein was more than 50-fold less active on its own than when supplemented with ovary lysate from hen1f00810 homozygous flies. It is speculated that the Ago2-RISC machinery is required for Hen1 function in flies, although the possibility cannot be excluded that the lysate contains a factor (e.g., a kinase) required for activating Hen1 (Horwich, 2007).
Modification of single-stranded siRNAs -- that is, those loaded in fully mature Ago2-RISC but not double-stranded siRNAs might allow cells to distinguish siRNAs loaded successfully into functional complexes from those that fail to assemble. For example, if a 3'-to-5' nuclease acts to degrade single-stranded siRNAs, 2'-O-methylation of single-stranded siRNAs in Ago2 RISC may protect them from destruction. Moreover, such a nuclease might trim the 3' end of piRNAs. 2'-O-methylation of the piRNA 3' terminus may occur only when the length of RNA extending beyond the Piwi-family protein is short enough to permit the simultaneous binding of the final ribose sugar to the active site of DmHen1 and the interaction of DmHen1 with the Piwi protein itself. Modification of the terminus of the trimmed piRNA would then block further 3'-to-5' trimming of the small RNA, generating its Piwi-, Aubergine-, or Ago3-specific length. The observation that piRNAs are shorter in hen1f00810 mutants supports this model (Horwich, 2007).
It is noted that all 2'-O-methyl-modified small RNAs identified thus far are associated with RISC complexes that efficiently cleave their RNA targets, i.e., Ago1-associated plant miRNAs, animal piRNAs, and Ago2-associated siRNAs in flies, whereas Drosophila miRNAs are typically both unmodified and associated with Ago1 RISC, which does not catalyze mRNA target cleavage in vivo. It is speculated that DmHen1 is recruited to RISC complexes containing single-stranded small silencing RNAs according to the identity of their Argonaute protein. This model predicts that DmHen1 will bind only to complexes containing fly Ago2 or the three fly Piwi proteins, Piwi, Aubergine, and Ago3, but not Ago1. Clearly, future experiments will need to test this hypothesis (Horwich, 2007).
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).
Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. The RNAi effector nuclease has been purified from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex, Argonaute2, is a homolog of genes that are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems (Hammond, 2001).
Posttranscriptional silencing phenomena have also been observed in plants (e.g., PTGS) and fungi (e.g., quelling): genetic studies indicate that these are likely to be mechanistically related to RNAi. Moreover, RNAi per se has been demonstrated in a variety of experimental systems, including insects, protozoans, and mammals. A synthesis of in vivo and in vitro experiments has led to a mechanistic model for RNAi/PTGS. Silencing is initiated by exposure of a cell to dsRNA. This 'trigger' may be introduced experimentally or may derive from endogenous sources such as viruses, transgenes, or cellular genes. Double-stranded RNAs are processed into discrete ~21- to 25-nucleotide (nt) RNA fragments known as siRNAs (small interfering RNAs). These small RNAs join a multicomponent nuclease complex, RISC, and guide that enzyme to its substrates through conventional base-pairing interactions. Recognition of mRNAs by RISC leads to their destruction (Hammond, 2001).
To date, mechanistic studies have approached RNAi/PTGS from two standpoints. Genetic studies have identified nearly a dozen genes that affect the dsRNA response. These include genes that encode putative nucleases (mut-7), helicases (qde-3, SDE3, mut-6), RNA-dependent RNA polymerases (e.g., ego-1, qde-1, SDE1/SGS2), and members of the Argonaute family (rde-1, qde-2, AGO1). Biochemical studies, carried out exclusively in extracts from Drosophila embryos and cultured cells, have identified enzymatic activities that are proposed to contribute to the interference process. However, links between biochemical and genetic studies of RNAi have yet to be made (Hammond, 2001 and references therein).
This study reports attempts to identify the proteins and RNAs that carry out RNAi in vitro as a step toward unifying biochemical and genetic data into a single mechanistic model. A ribonuclease III family enzyme, Dicer, is a candidate for processing long dsRNA silencing triggers into ~22-nt siRNAs. A requirement for Dicer in RNAi in vivo has been demonstrated in C. elegans. This study reports the biochemical purification of RISC, the effector nuclease of RNAi, and the identification of one subunit of this enzyme. This protein is a member of the Argonaute family, which has been linked to RNAi through genetic studies in several experimental systems (Hammond, 2001).
RNA interference can be provoked in cultured Drosophila S2 cells by transfection with dsRNA, or indeed by simply adding dsRNA to the culture media. Extracts from such cells contain a nuclease complex, RISC, that specifically degrades mRNAs that are homologous to the dsRNA trigger. The hypothesis that this nuclease constitutes the effector activity of RNAi is strengthened by the observation that RISC cofractionates with ~22-nt RNAs that are derived directly from the silencing trigger. Furthermore, this nuclease contains an essential nucleic acid subunit, which is presumably a siRNA (Hammond, 2001).
A biochemical fractionation protocol has been developed that permits the purification of RISC to near-homogeneity. RISC is bound to ribosomes in cell-free extracts; however, the biological relevance of this association remains to be established. Ribosomes can be concentrated from S2 lysates by high-speed centrifugation, and soluble RISC can be recovered from the ribosome pellet by extraction with high concentrations of salt (Hammond, 2001).
Size fractionation of soluble RISC yielded a single peak of sequence-specific nuclease activity. Thus, a single complex contains all the activities and information needed to identify and degrade cognate mRNAs. The large size of this complex (~500 kD) is consistent with its being composed of several subunits, which, according to previous studies, comprise both RNA and protein. A series of additional chromatographic steps were developed that yielded a fraction with a sequence-specific nuclease activity that was purified ~1:10,000 from the crude extract (Hammond, 2001).
Analysis of fractions from the hydroxyapatite column by SDS-polyacrylamide gel electrophoresis (PAGE) indicates that the complex has not been purified to complete homogeneity; however, several proteins clearly cofractionate with the active RISC fraction. Candidate proteins were excised from the gel and microsequenced using tandem mass spectroscopy. Two of four bands failed to produce protein sequence. However, numerous peptides were obtained from bands of ~87 and ~130 kD that matched a single Drosophila gene. Database and domain searches identified this peptide as a homolog of rde-1, a member of the Argonaute gene family, which is essential for RNAi in C. elegans. This gene has been named Argonaute2 (AGO2, Flybase annotation number CG7439) because of the prior assignment of Argonaute1 to another gene in the Drosophila genome. Although the Drosophila genome contains at least four Argonaute family members -- AGO1, AGO2, Piwi, and Sting, only AGO2 has been idenfied as a component of RISC in S2 cells. However, the possibility that other Drosophila Argonaute family members join the RISC complex in specific tissues or at specific times during development cannot be excluded (Hammond, 2001).
To verify the presence of AGO2 in RISC, AGO2-specific antibodies were generated. Western blotting of chromatography column fractions with affinity-purified anti-AGO2 shows precise cofractionation of a ~130-kD AGO2 protein and the active RISC fraction through each purification step. In addition, the association between AGO2 and other components of RISC, the siRNAs, was tested. A version of AGO2 was constructed that was tagged at its NH2-terminus with both a T7 epitope and polyhistidine. This was expressed in cells in which RNAi had been induced against firefly luciferase. Tagged AGO2 protein cofractionates with endogenous AGO2, and with the active RISC fraction, in the 500-kD size range. RISC was affinity-purified from cell extracts on a polyhistidine-binding resin. Analysis of the imidazole elution profile from this column by Western blotting with a T7 antiserum and by Northern blotting with a luciferase probe indicates cofractionation of the tagged AGO2 and 22-nt siRNAs. Considered together, these data strongly support the hypothesis that AGO2 is a component of RISC (Hammond, 2001).
To test whether AGO2 is essential for RNAi in Drosophila S2 cells, RNA interference was used to suppress endogenous AGO2. Treatment of S2 cells with either of two different ~1000-nt dsRNAs homologous to AGO2 reduces the levels of this protein by a factor of >10. The ability of these cells to carry out RNAi was assessed by transfection with a mixture of firefly and Renilla luciferase expression plasmids (as an internal control) in combination with either a control dsRNA (green fluorescent protein, GFP) or a firefly luciferase dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence an exogenous reporter by RNAi (Hammond, 2001).
The biochemical function of Argonaute family members is completely unknown. However, one domain of this protein, the PAZ domain, is shared with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs. The possibility that Dicer and AGO2 might physically interact, perhaps through their shared PAZ domains, was therefore considered. Indeed, endogenous AGO2 can be coimmunoprecipitated with an epitope-tagged version of Dicer protein from transfected S2 cells. Dicer and RISC are biochemically separable, and none of the purified RISC fractions is able to process dsRNA into 22-nt fragments. One possibility is that Dicer is indeed a component of RISC but fails to process dsRNA when present in this complex. However, the current model is that the interaction between AGO2 and Dicer facilitates the incorporation of siRNAs into RISC complexes, which ultimately dissociate from Dicer and target cognate mRNAs for destruction (Hammond, 2001).
Previous genetic studies in three organisms have indicated that Argonaute family members are essential for RNAi/PTGS. The first link between Argonaute proteins and RNAi was shown by the isolation of C. elegans rde-1 in a screen for RNAi-deficient mutants. In Neurospora, another member of the Argonaute family, QDE-2, emerged from a selection of mutants that were defective in a transgene cosuppression phenomenon, termed 'quelling'. The founding member of this family (AGO1) was first identified in Arabidopsis in a screen for mutants with aberrant leaf morphology. Subsequently, ago1 was re-isolated in a screen for plants that were defective in transgene cosuppression (Hammond, 2001).
Argonaute proteins are typically members of multigene families. In Drosophila there are four annotated genes: Sting, Piwi, AGO1, and AGO2. Mutations in three family members (ago1, piwi, and sting) have previously been studied. Piwi is required for maintenance of cell proliferation in both the male and female germ line, and sting mutations produce spermatid defects and male sterility. Ago1 was identified in a screen for mutations in the wingless pathway, and null mutations in this gene cause defects in neurological development (Hammond, 2001 and references therein).
Thus, Argonaute family members have been linked both to gene silencing phenomena and to the control of development in diverse species. The critical question is whether these two roles of Argonaute proteins are mechanistically related. It is already clear that RNAi-related silencing pathways can control the activity of endogenous genetic elements (e.g., transposons). The possibility also exists that these same pathways may control the expression of endogenous protein-coding genes that regulate development. An answer to this question is likely to emerge both from further genetic studies of RNAi pathways and from a search for endogenous targets of RISC that may be identified via its internal RNA guides to substrate selection (Hammond, 2001).
RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. This study reports biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, homology was found between the PIWI domain of Ago-2 and endonuclease V and potential active-site amino acid residues within the PIWI domain of Ago-2 were identified (Rand, 2004).
RNA interference is the phenomenon in which long double-stranded (ds)RNA is able to silence cognate gene expression. First, the dsRNA is processed into small interfering RNA (siRNA) by a Dicer enzyme. In Drosophila cells, siRNA is retained by Dicer-2 and an associated protein, called R2D2. The Dicer-2-R2D2 complex is then able to facilitate the loading of siRNA onto a complex known as the RNA-induced silencing complex (RISC). The RISC-bound siRNA strand acts as a guide to identify mRNA targets with complementary sequence for nucleolytic cleavage. RISC activity is defined simply as siRNA-guided, site-specific cleavage of an mRNA target (Rand, 2004 and references therein).
The guide RNA in RISC is held by the PAZ domain of Argonaute 2. This protein was recognized when RNA interference-defective mutants were found in Caenorhabditis elegans. Ago-2 was also found to correlate with partially purified RISC activity from Drosophila and human cells. Furthermore, siRNAs were demonstrated to copurify with Ago-containing complexes. Finally, the PAZ domain of Ago-2 was determined to be an siRNA-binding domain by structural studies (Rand, 2004 and references therein).
Several characteristic features of RISC nucleolytic activity have been determined. RISC cleaves the target mRNA strand between positions 10 and 11, counted from the 5' to 3' end of the guide RNA. These cleavage products have a 5' phosphate and a 3' hydroxyl group. Finally, RISC activity is magnesium-dependent. Although a long list of proteins have been proposed to be RISC components, none has been identified that accounts for the nuclease activity of RISC (Rand, 2004 and references therein).
A biochemical fractionation approach was used to purify the RISC nuclease activity to homogeneity from Drosophila Schnieder 2 (S2) cells. RISC activity was found to be generated by a single protein, Ago-2. A sequence information comparison tool was used to predict that the PIWI domain of Ago-2 is similar to the endonuclease V in sequence and secondary structure, suggesting that the PIWI domain provides the missing nuclease activity of RISC. An alignment of PIWI domain with several endonucleases was used to predict three residues, aspartate 965 (GADVT), glutamate 1016 (TLEHL), and aspartate 1037 (YRDGV), as being magnesium-coordinating residues at the catalytic center of Ago-2 nuclease. The corresponding two aspartate residues have also been recognized as part of the nuclease active site in mammalian and archaebacterium Pyrococcus furiosus Ago-2 by structural and mutagenesis studies (Rand, 2004).
To study RISC activity, let-7 siRNA was added to S-100 extracts prepared from Drosophila S2 cells and siRNA-dependent cleavage of a complementary mRNA was generated. The sensitivity of RISC activity to high salt (up to 1 M) was analyzed both before and after siRNA addition. High salt exposure after siRNA addition reversibly masked the ability of RISC to cleave target substrate. After the salt was removed by dialysis, full RISC activity was recovered. However, upon exposure of S2 cell extracts to high salt prior to siRNA loading, RISC activity was irreversibly lost; it could not be recovered even after the salt was completely dialyzed away. The difference in salt sensitivity suggested that the assembly of RISC (by addition of siRNA to naive extracts) involves a component (or components) with a molecular conformation that is irreversibly damaged upon salt addition, and it also suggested that the salt-labile, functioning conformation is upstream of the siRNA-loaded RISC. The salt sensitivity made it difficult to purify the components needed for de novo RISC assembly. However, resistance of preloaded RISC to high salt exposure makes it a better subject for protein-purification procedures. Therefore, a procedure was developed for the purification of preloaded RISC nuclease activity to homogeneity (Rand, 2004).
The purification procedure began with the addition of a 3'-biotinylated siRNA to 200 ml of S-100 extract (1 g of total protein) from S2 cells. After incubation at room temperature for 120 min to load the siRNA into RISC, the RISC activity was collected by centrifugation at 200,000 x g. The pelleted RISC activity was then solubilized by extraction with 400 mM potassium acetate. After addition of ammonium sulfate to 20% saturation, the 100,000 x g supernatant, containing RISC activity, was loaded onto a phenyl-Sepharose column and eluted with a decreasing ammonium sulfate/potassium acetate gradient over 10 column volumes. The RISC activity was eluted in a sharp peak around fractions 19-22. The active fractions were pooled, dialyzed to 200 mM KOAc, and loaded onto a Q-Sepharose column. The RISC activity that flowed through this column was subsequently loaded onto a heparin agarose column, and the RISC activity was eluted by a 15%-100% step of 1 M KOAc. The eluate was dialyzed buffer, and the RISC activity was pulled down by using streptavidin-conjugated magnetic beads by overnight incubation with rotation at 4°C in the presence of 1% Triton X-100. The RISC activity pulled down by the beads was then split. An aliquot of 10% of the beads was used for the RISC assay, and the remaining 90% was used for protein identification. Before assaying, the beads were coated with Denhardt's reagent to keep the mRNA substrate from sticking to the beads, a step critical for assaying RISC activity directly on beads. The RISC activity was enriched and pulled down by the beads when biotin-labeled, but not normal, siRNA was used. Because the RISC activity was resistant to high salt, another five washes of high salt were added. The final high-salt wash did not affect the RISC activity on the beads, and the target RNA was completely processed (Rand, 2004).
Protein purifications are normally finished by running an SDS gel electrophoresis to identify correlating bands. However, proteins with unusual sizes, extremely positive charges, low abundance, or those that are poor substrates for staining (for instance, with silver stain) may be lost during this step. All of these potential pitfalls were avoided by directly digesting all of the proteins on the beads with trypsin and subjecting all resulting peptides directly to MS. Interestingly, if the beads were not subjected to the final high-salt wash, many proteins (including previously identified RISC component Ago-2) were identified. Most of other proteins were ribosomal and other RNA-binding proteins. However, when the beads were subjected to high-salt wash before digestion with trypsin, all of the 33 peptides recovered were from Ago-2. Importantly, not a single peptide from any other protein was detected under this condition. Also, all of the nonspecific proteins that were pulled down by the bead when normal siRNA was used for RISC assembly were washed away by high salt. Therefore, it was conclude that one protein, Ago-2, composes the core RISC nuclease activity (Rand, 2004).
Previous attempts to purify RISC nuclease from human and Drosophila systems have identified Ago-2 as well as several other proteins, including dFXMR, VIG, and tudor-SN, all associated with RISC. However, the roles of these proteins in the fundamental activity of RISC (siRNA-guided, site-specific endonucleolytic cleavage) are still unclear. RISC activity has also been purified to near homogeneity from HeLa cell extract. A correlation between only two proteins, eIF2C2 and eIF2C1 (two Ago family members), and RISC activity was seen. This work is especially important because it rules out many of the previously identified RISC-associated proteins as being required for RISC activity, and it suggests that RISC activity might be provided by an Ago family protein alone. It was reasoned that purification of RISC activity to homogeneity judged by the most rigorous standard would provide answers to what the actual composition of the RISC complex was and this led to identification of the RISC nuclease. Surprisingly, Ago-2 is the only detected protein with the recovered peptides covering most of the protein. This result provides strong evidence that Ago-2 alone is sufficient for siRNA-loaded RISC activity (Rand, 2004).
This finding was surprising because there had been no previous indication that Ago-2 had any nuclease domain. After a careful bioinformatics analysis based on sequence and predicted secondary structure, it was realized that the PIWI domain of Ago-2 shows similarity to endonuclease V. This alignment predicts three active-site residues. Two recently published articles reported a nearly identical conclusion from two different experimental systems. In the first article, the structure for the Ago protein from archaebacterium P. furiosus was solved (Song, 2004). The structure revealed that the PIWI domain fold is similar to RNase H, with two conserved active-site residues that fit perfectly with the bioinformatics-based prediction of the active site of PIWI to endonuclease V. Although no structure is available for endonuclease V, sequence analysis strongly suggests that it should possess an RNase H-like fold. RNase H hydrolyzes RNA from an RNA-DNA hybrid ds molecule, whereas endonuclease V nicks DNA near chemically damaged sites on dsDNA substrates. Endonuclease V also has the ability to cleave undamaged single-stranded DNA. Both proteins are identical to the RISC endonuclease in terms of magnesium dependence and the molecular ends produced after cleavage (Rand, 2004).
In the second article, site-directed mutagenesis on two of the structurally predicted and alignment-predicted active-site residues in mammalian Ago-2 protein abolished RISC activity when the mutant proteins were expressed in the Ago-2 knockout mouse embryonic fibroblasts (Liu, 2004). The third Mg2+ coordination site has not yet been experimentally determined. When considering the biochemical purification and the published structure analysis of Ago-2 together, it is clear the siRNA-Ago-2 complex alone is sufficient for RISC activity (siRNA-guided, site-specific cleavage of mRNA targets), with the PIWI domain of Ago-2 functioning as the nuclease (Rand, 2004).
Although only one Dicer enzyme is found in C. 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. 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 to be DCR-2 and a previously unknown protein (Flybase CG7138 or R2D2), respectively. R2D2 bears 20.9% identity and 33.4% similarity to the C. elegans RNAi protein RDE-4, which also contains tandem dsRNA-binding domains and interacts with Dicer (Liu, 2003).
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. 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, streptavidin beads precipitated AGO2 protein only when biotinylated siRNA was used, suggesting that the siRNA RISC activity was specific. 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).
How does the RISC-loading complex (RLC), with the Dcr-2/R2D2 heterodimer positioned asymmetrically on the siRNA, progress to the RISC? Argonaute 2 is a ~130-kD protein that is a core component of the RISC and is required for siRNA unwinding. An ~130-kD protein was crosslinked to siRNA when the guide strand contained 5-iodouracil at p20. The ~130-kD protein was photocrosslinked only to the guide strand of the siRNA, which suggests that this protein is a component of the RISC. The ~130-kD protein was immunoprecipitated with antibodies to Ago2 but not to Ago1 and was not observed in embryos lacking both maternal and zygotic Ago2 (ago2414). Thus, the ~130-kD protein is Ago2. When R2D2 and Ago2 were photocrosslinked to siRNAs that contain 5-iodouracil at p20 of the passenger or the guide strand, R2D2 is bound to the 3' end of the guide strand and Dcr-2 to the 3' end of the passenger strand at early times in the reaction. Later, binding of R2D2 and Dcr-2 decrease concurrently, accompanied by a corresponding increase in binding of Ago2 to the 3' end of the guide strand. In ago2414 lysates, R2D2 binding to the 3' end of the guide strand and Dcr-2 binding to the 3' end of the passenger strand did not decrease with time; this finding suggests that binding of Ago2 facilitates the release of the heterodimer from siRNA (Tomari, 2004).
The siRNA bound by Ago2 is single-stranded, because Ago2, when photocrosslinked to siRNA, was captured by a tethered 2'-O-methyl oligonucleotide complementary to the siRNA guide strand. R2D2 is not captured by the 2'-O-methyl oligonucleotide, but is instead recovered in the supernatant, consistent with R2D2 binding of double-stranded siRNA (Tomari, 2004).
The data suggest a model for RISC assembly. Initially, R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RLC. As siRNA unwinding proceeds, the heterodimer is exchanged for Ago2, the core component of the RISC. Indeed, single-stranded siRNA was not detected in the RLC assembled in ago2414 lysate. It is hypothesized that unwinding occurs only when Ago2 is available, so that siRNA in the RLC is unwound only when the RISC can be assembled (Tomari, 2004).
In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).
The identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).
Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).
The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).
The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).
RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms (Li, 2002).
Focus was placed on the flock house virus (FHV) because its B2 gene shares key features, but not sequence similarity, with the plant cucumoviral 2b gene of cucumber mosaic virus (CMV), which encodes a known group of silencing suppressors. Both open reading frame (ORF) 2b and B2 overlap the carboxyl terminal region and occupy the +1 reading frame of the ORF encoding the viral RNA-dependent RNA polymerase and are translated in vivo by a subgenomic mRNA (Li, 2002).
The FHV B2 protein indeed exhibited a potent silencing-suppression activity in the Agrobacterium co-infiltration assay, established in transgenic plants that express green fluorescent protein (GFP). Transient B2 expression prevented RNA silencing of the GFP transgene, leading to a strong and prolonged green fluorescence examined under ultraviolet (UV) illumination, similar to suppression by the cucumoviral 2b proteins. In contrast, a broad red fluorescent zone surrounding the infiltrated patch becomes clearly visible 6 days after infiltration, when the co-infiltrated transgene directs translation of neither 2b nor B2 (Li, 2002).
RNA blot hybridizations confirmed that expression of either protein is associated with high accumulation levels of the GFP mRNA. In addition, the GFP-specific siRNAs, a hallmark of RNA silencing, remain at extremely low levels in the leaves where there is expression of either B2 or 2b. B2 is able to functionally substitute for 2b of cucumber mosaic virus in whole plant infections, as found previously for a CMV 2b homolog. B2 suppression of RNA silencing in plants explains why FHV is able to overcome the RNA silencing defense and establish systemic infections in transgenic plants that express a plant viral protein that facilitates virus cell-to-cell movement (Li, 2002).
The finding that an FHV-encoded protein suppresses RNA silencing in plants suggests a role for RNA silencing in FHV infections of animal hosts. FHV belongs to the Nodaviridae family, members of which naturally infect vertebrate and invertebrate hosts, and Drosophila cells support complete infection cycles of FHV. Infection of Drosophila S2 cells with FHV virions resulted in a rapid appearance of the FHV-specific siRNAs of both positive and negative polarities. Accumulation of the siRNAs trailed that of FHV genomic and subgenomic RNAs, which suggests that the decreased accumulation of FHV RNAs at later stages of FHV infection may be caused by an FHV-specific RNA silencing (Li, 2002).
To investigate this possibility, a full-length FHV RNA1 cDNA clone (pRNA1) was constructed, which, after transfection into S2 cells, directed RNA1 self-replication and transcription of RNA3, the subgenomic mRNA for B2. Depleting the mRNA for Argonaute2 (AGO2) by RNAi, an essential component of the RISC complex, led to a pronounced increase (two- to three-fold) in the accumulation of FHV RNAs 1 and 3, indicating that a functional RNA-silencing pathway naturally restricted FHV accumulation in the host cells. Furthermore, co-transfection of pRNA1 with a dsRNA targeting the 3'-terminal 500 nucleotides of FHV RNA1 completely prevented the accumulation of intact FHV RNA1 in S2 cells. These results collectively demonstrate that FHV is both an initiator and a target of RNA silencing in this animal host (Li, 2002).
Further studies showed that B2 was essential for FHV accumulation in Drosophila cells, which is in contrast to a previous study carried out in nonhost mammalian cells. A B2-knockout mutant of FHV RNA1, referred to as RNA1-Delta B2, which contains point mutations that convert the first and 58th codons of the B2 ORF into serine and stop codons, respectively, failed to accumulate to detectable levels after transfection into S2 cells. This defect was partially trans-complemented (up to 10% of the wild-type level) by co-transfection of a plasmid expressing either B2 or a His-tagged B2. Expression of the His-tagged B2 from the co-transfected plasmid was detected in S2 cells by Western blot analysis using an antibody recognizing the His tag. Reverse transcription-polymerase chain reaction and sequencing revealed that the introduced mutations were stably maintained in the progeny FHV RNAs extracted from infected cells, indicating that B2 was indeed expressed from the co-transfected plasmid rather than from a revertant RNA1 (Li, 2002).
Accumulation of RNA1-Delta B2 in S2 cells was efficiently rescued, up to 50% of the wild-type level, by co-transfection with the AGO2 dsRNAs, either singly or in combination. However, co-transfection with dsRNAs targeting mRNAs of the two Drosophila Dicer genes was not effective under the same conditions. This is possibly due to a more efficient mRNA depletion by RNAi for AGO2 than for Dicer, which is required to process the input dsRNA. Notably, the level of complementation by RNAi of AGO2 was higher than that achieved by the B2-expressing plasmid, although this level was still achieved less efficiently than B2 expression from wild-type RNA1. Therefore, in the absence of B2 expression, FHV RNAs 1 and 3 accumulate to substantial levels when the RISC complex is disrupted by AGO2 depletion. These data confirm the finding that B2 is not required for RNA1 self-replication and indicate that the essential function of B2 for FHV infection of the S2 host cells observed in this study is to suppress RNA silencing that targets FHV RNAs for degradation. Thus, the same protein blocks RNA silencing in both animals and plants, providing the first experimental evidence for a highly conserved RNA silencing pathway in different kingdoms (Li, 2002).
It is known that RNA silencing operates in animals, including mammals. This work demonstrates that infection of Drosophila cells with an RNA virus triggers strong virus RNA silencing and that the same virus is equipped with an effective silencing suppressor essential for infection. These data provide direct evidence that RNA silencing naturally acts as an adaptive antiviral defense in animal cells. The specificity mechanism of this adaptive defense is based on nucleic acid base pairing between siRNA and its target RNA and thus is distinct from cellular and humoral adaptive immunity based on peptide recognition. A prediction from this work is that heterologous sequences inserted into a replicating virus genome will lead to the production of a population of siRNAs capable of silencing other viral and cellular RNAs in trans that are homologous to the insert. Indeed, recent studies have shown that viral sequences inserted in alphavirus vectors give rise to virus resistance in mosquitoes, which is dependent on the inserted RNA sequence rather than on its protein product. It will be of interest to determine if RNA silencing also plays a role in observed protection against mammalian viruses, derived similarly from heterologous expression of RNA sequences from a replicating RNA virus vector (Li, 2002).
Homology-dependent RNA silencing occurs in many eukaryotic cells. Nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Evidence is presented to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway; the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. RNAi also functions as a natural antiviral immunity in mosquito cells. Vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. The double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells (Li, 2004).
The biochemical framework of the core RNA silencing pathway is based mostly on the experimental induction of RNA silencing by exogenous dsRNA, commonly referred to as RNAi. Thus, an investigation was carried out to see whether B2 of FHV, known to inhibit RNA silencing induced by viral RNA replication, also suppresses RNAi. To this end, Drosophila cells were transfected with a plasmid encoding either GFP or B2 fused with GFP (B2-GFP) under the control of an inducible promoter, and 1 day after induction of expression of GFP or B2-GFP, dsRNA targeting the GFP coding sequence was introduced. B2-GFP remained active in suppressing virus RNA silencing because cotransfection with either pB2-GFP or pB2 rescued the in vivo accumulation of the self-replicating FHV RNA1 mutant carrying an untranslatable B2, called FR1-DeltaB2. Suppression of AGO2 expression by the specific dsRNA also rescued the accumulation of FR1-DeltaB2 RNAs. In the GFP RNAi assay, targeting of the B2-GFP mRNA by RNAi occurred in cells that were also expressing B2-GFP. Northern blot hybridizations found that accumulation of GFP mRNA was significantly reduced 2 days after GFP dsRNA was introduced as compared with treatment with lacZ dsRNA, indicating an effective RNAi against GFP mRNA by this sequential transfection protocol. However, no obvious difference in the accumulation of B2-GFP mRNA was observed between cells treated with either GFP or lacZ dsRNA. Thus, the specific degradation of mRNA targeted by a homologous dsRNA did not occur in Drosophila cells expressing the B2 fusion protein, establishing FHV B2 as a viral suppressor of RNAi in an animal system (Li, 2004).
When GFP dsRNA was cotransfected with pB2-GFP, however, B2-GFP mRNA was effectively destroyed by RNAi. Thus, RNAi suppression requires B2 expression before dsRNA is introduced into cells to initiate RNAi, suggesting that B2 suppression may occur before the production or RISC loading of siRNAs. Collectively, these data show that RNA silencing triggered by either dsRNA or viral RNA replication not only is AGO2-dependent, but also is sensitive to B2 suppression, providing compelling evidence that the RNA silencing antiviral immunity detected in Drosophila cells is mediated by the RNAi pathway (Li, 2004).
Whether the RNA silencing antiviral response is elicited when Drosophila cells are challenged with another virus was investigated. NoV is the only member of the Alphanodavirus genus that can lethally infect both insects and mammals. However, the sequence identity between the encoded proteins of NoV and FHV is either low (44% for the viral RdRP) or minimal (<19% for B2). A full-length NoV RNA1 cDNA clone (pNR1) was constructed encoding a self-replicating RNA. After transfection into the Drosophila S2 cells and transcriptional induction of the viral cDNA, pNR1 directed RNA1 self-replication and transcription of RNA3, the subgenomic mRNA for B2. A point mutation, as in NR1-DeltaB1, that abolished translation of the overlapping B1 ORF from RNA3, corresponding to the C-terminal portion of the viral RdRP, had no effect on the accumulation of either RNA1 or RNA3. However, a B2-knockout mutant, referred to as NR1-DeltaB2, was hardly detectable in transfected Drosophila cells by Northern blot hybridizations. NR1-DeltaB2 contained point mutations that prevented B2 translation but did not change any amino acid in the -1 reading frame that codes for RdRP. The defect of NR1-DeltaB2 accumulation in WT Drosophila cells was complemented in trans by cotransfection of a plasmid expressing the NoV B2 protein (nB2), indicating that nB2 is required for NoV RNA accumulation (Li, 2004).
Several lines of evidence indicate that NoV RNAs were targeted for silencing by the AGO2-dependent RNAi pathway in Drosophila cells and that nB2 suppressed the RNAi antiviral response to ensure successful NoV RNA replication and transcription. (1) The FHV B2 protein (fB2), shown to suppress RNAi, rescued accumulation of NR1-DeltaB2 in Drosophila cells. (2) Depleting RISC by either dsRNA or siRNA targeting AGO2 efficiently rescued accumulation of NR1-DeltaB2. Such a rescue was not observed by cotransfection with an unrelated lacZ siRNA or dsRNA. These results indicate that rescue of NR1-DeltaB2 by cotransfection of dsRNA and siRNA targeting AGO2 is caused by a specific AGO2 depletion, rather than a nonspecific effect of dsRNA. Similar specific rescue of FR1-DeltaB2 in Drosophila cells by either the dsRNA or siRNA targeting AGO2, but not by the dsRNA and siRNA targeting lacZ, was also observed. In addition, nB2 suppressed RNA silencing targeted against both FR1-DeltaB2 in transfected Drosophila cells and a GFP transgene in transgenic plants (Li, 2004).
Remarkably, an effective suppression was found of RSAR targeting FR1-DeltaB2 in Drosophila by the tombusvirus 19-kDa protein (p19), but not after it was truncated . This finding demonstrates cross-kingdom suppression of RNA silencing in an animal system by a plant viral suppressor. Together with the observations that the B2 proteins encoded by two animal nodaviruses suppress RNA silencing in both Drosophila and tobacco plants, these results show that the RSAR mechanism is conserved between the plant and animal kingdoms (Li, 2004).
Because the genome of the mosquito A. gambiae, which transmits both malaria and viruses, encodes a functional RNAi pathway similar to Drosophila, whether RNAi also protects A. gambiae against NoV was investigated. Self-replication of NoV RNA1 and transcription of RNA3 were detected in cells after transfection with pONR1, which contained the NoV RNA1 cDNA under the control of the OplE2 promoter. pNR1 failed to initiate RNA replication in A. gambiae cells, possibly because of a lack of transcriptional induction. Neither RNA1 replication nor RNA3 transcription was detected in A. gambiae cells transfected with pONR1-DeltaB2, which encoded the B2-knockout mutant of NoV RNA1. However, the defect was rescued by cotransfection with either a plasmid expressing a B2 or a dsRNA corresponding to mRNA of the A. gambiae AGO2. The rescue of ONR1-DeltaB2 was also observed by cotransfection with an siRNA targeting AGO2, although with an efficiency ~50% lower than the long dsRNA. ONR1-DeltaB2 rescue by AGO2 depletion was specific, since cotransfection with dsRNA to neither lacZ nor AGO1 mRNA was effective. Thus, as found in Drosophila, a self-replicative NoV RNA also triggered the RNAi antiviral response in A. gambiae cells, which is both AGO2 dependent and sensitive to B2 suppression, establishing this immunity pathway in two different insect cell lines. This finding opens up the possibility of targeting this pathway to prevent transmission of mosquito-borne human viral diseases such as dengue (Li, 2004).
To facilitate screening for new animal RNAi suppressors, the coding sequence of GFP was fused in-frame with the start of the B2 ORF of FHV RNA1 to yield pFR1gfp. pFR1gfp was essentially a B2-knockout mutant carrying a visual marker because only the first 23 aa of the 106-aa residue fB2 were translated in the fusion protein. Indeed, GFP cells were not visible after pFR1gfp was transfected alone but became abundant when it was cotransfected with a B2-expressing plasmid. GFP expression was detected also when pFR1gfp was cotransfected with the AGO2 dsRNA. Replication and accumulation of FR1gfp in S2 cells cotransfected with either the AGO2 dsRNA or the fB2-expressing plasmid were confirmed by Northern blot detection of both positive- and negative-strand viral RNAs. Thus, FR1gfp was defective in suppressing RSAR in Drosophila cells as was FR1-DeltaB2. Importantly, B2 suppression of the Drosophila RNA silencing response targeting either FR1gfp or FR1-DeltaB2 did not require prior B2 expression, which was found to be necessary for RNA silencing induced by dsRNA. This finding is consistent with the natural situation in which the expression of B2 after viral RNA replication but early in infection is sufficient to allow productive FHV infection and establishes an easy cell-based assay for the identification of animal RNAi suppressors, simply by detection of GFP after cotransfection of pFR1gfp with a plasmid expressing a candidate protein (Li, 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).
In the Drosophila and mammalian RNA interference pathways, siRNAs direct the protein Argonaute2 (Ago2) to cleave corresponding mRNA targets, silencing their expression. Ago2 is the catalytic component of the RNAi enzyme complex, RISC. For each siRNA duplex, only one strand, the guide, is assembled into the active RISC; the other strand, the passenger, is destroyed. An ATP-dependent helicase has been proposed first to separate the two siRNA strands, then the resulting single-stranded guide is thought to bind Ago2. This study shows that Ago2 instead directly receives the double-stranded siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand, thereby liberating the single-stranded guide. For siRNAs, virtually all RISC is assembled through this cleavage-assisted mechanism. In contrast, passenger-strand cleavage is not important for the incorporation of miRNAs that derive from mismatched duplexes (Matranga, 2005).
The data suggest a new model for Ago2 RISC assembly in which siRNAs are initially loaded into Ago2 as duplexes, and then Ago2 cleaves the passenger strand of the siRNA, facilitating its displacement and leaving the siRNA guide strand bound stably to Ago2. In support of this cleavage-assisted assembly model, it was found that the passenger strand is cleaved during RISC assembly. Both the position of the cleavage site on the passenger strand and the stereospecificity of inhibition by phosphorothioates are diagnostic of siRNA-directed, Ago2-catalyzed cleavage. Both Ago2 and the RISC-loading complex (RLC) are required for this cleavage, and cleavage occurs before the passenger strand has dissociated from the guide strand -- before the formation of active RISC (Matranga, 2005).
A cleavage-assisted assembly mechanism is also consistent with the finding that early (5 min) in assembly, RISC contains considerable amounts of double-stranded siRNA. When the asymmetry rules for siRNA loading were first uncovered, they evoked the idea of a nonprocessive helicase that separated the two strands of the duplex, starting from the end that was less stably paired. However, subsequent studies showed that the R2D2/Dcr-2 heterodimer, the core of the RLC, binds asymmetrically to double-stranded siRNA, suggesting that a nonprocessive helicase does not sense siRNA thermodynamic asymmetry during RISC loading. Unlike Ago2 loaded via the RISC assembly pathway, affinity-purified or recombinant Ago2 can only be programmed with single-, not double-stranded siRNA, suggesting that the essential function of the RLC is to facilitate the directional loading of a double-stranded siRNA into Ago2. The idea that Ago2-mediated passenger-strand cleavage triggers siRNA strand dissociation also accounts for the previous observation that one passenger strand appears to be destroyed for every cycle of assembly of a guide strand into target-cleaving RISC. Passenger-strand cleavage would strongly reinforce siRNA functional asymmetry by coupling passenger-strand destruction to RISC assembly (Matranga, 2005).
Blocking Ago2-mediated passenger-strand cleavage by substituting a phosphorothioate for the scissile phosphate revealed a slower bypass mechanism that dissociates the two siRNA strands. Of course, phosphorothioate-substituted siRNAs do not occur in nature, but miRNA/miRNA* duplexes do often contain central mismatches predicted to block cleavage of the miRNA* strand, the analog of the siRNA passenger strand. Most, if not all, miRNAs are efficiently loaded into Ago2 in cultured human cells, and miR-127, miR-136, miR-196, miR-431, miR-433, and miR-434 are known to cleave their targets in vivo and are therefore presumed to function in an Ago2-containing RIS CMatranga, 2005).
It is envisioned that in the bypass pathway -- as in the cleavage-assisted pathway -- Argonaute proteins are loaded with double- rather than single-stranded siRNAs. Dissociation of the full-length passenger strand would then require breaking its interaction with the 'seed' region of the guide strand (nucleotides 2-7), a region proposed to mediate miRNA target pairing. For metazoan miRNAs, conserved Watson-Crick pairing to the seed is necessary and sufficient for accurate target prediction. Similarly, Watson-Crick pairing to the seed can be sufficient for miRNA-mediated repression, and pairing to this 5' region of the guide strand makes a far greater contribution to target binding affinity than does pairing to the 3' end. The tight binding to the 5' portion of the guide strand is what presumably prevents an appreciable amount of a standard siRNA duplex from being assembled into Ago2 through the bypass mechanism. In contrast, when presented to Ago2 as a miR-1/miR-1* duplex, miR-1 loads efficiently without need for passenger-strand cleavage, because a Watson-Crick pairing to the miRNA seed is disrupted. Indeed, far less disruption of seed pairing might be sufficient to enable the bypass mechanism to begin to play a substantial role, as hinted by results using the miR-1 'GU' siRNA, which has a single G:U wobble disrupting perfect Watson-Crick pairing to the seed. Passenger-strand cleavage appeared less important for this duplex than for the standard siRNA duplex, a result consistent with computational and experimental studies of miRNA target specificity imply that single G:U wobble pairs in the seed disproportionately perturb small RNA binding (Matranga, 2005).
In addition to loading miRNAs, the bypass mechanism might be used to load siRNAs into Argonaute proteins that have lost their catalytic amino acids, such as human Ago1, Ago3, and Ago4. Conversely, Arabidopsis thaliana Argonaute1, a miRNA-guided plant Argonaute protein with a functional endonuclease domain (Baumberger and Baulcombe, 2005 and Qi, 2005), might be loaded by a passenger-strand cleavage-assisted pathway. Although human Ago1 and Ago3 can bind standard siRNAs, endogenous Ago1 and Ago3 cannot support siRNA-directed RNAi in Ago2-knockout mouse embryonic fibroblasts. The function of Ago1, Ago3, and Ago4-containing RISC is not yet known. Perhaps the capacity for loading small RNAs via the cleavage-assisted pathway confers a degree of specificity to the function of different silencing complexes, with Ago2 able to use a broader range of small RNAs than those Argonaute proteins incapable of RNA cleavage (Matranga, 2005).
The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC (Rand, 2005).
Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ('Slicer') activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here it is shown that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. Upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as 'Slicer' of the passenger strand of the siRNA duplex (Miyoshi, 2005).
RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, mRNA expression profiles were examined in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. Cells depleted of the miRNA-processing enzyme Drosha were also examined. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought (Rehwinkel, 2006).
Using microarray analysis of Drosophila cells depleted of Drosha and Argonaute proteins, this study shows that transcripts whose levels are likely to be directly regulated by silencing pathways (up-regulated transcripts) represent less than 20% of the Drosophila S2 cell transcriptome. Computational predictions of miRNA targets indicate that more than 30% of the transcriptome is targeted by miRNAs. There are several possible explanations for these seemingly contradictory observations. First, it was shown that not all authentic targets change levels in a detectable manner. This indicates that although microarrays are a valuable tool to identify miRNA targets, many targets may escape detection using this approach. Second, some miRNAs and targets are expressed in a tissue-specific manner, so it is likely that only a subset of miRNA/target pairs is expressed in S2 cells. Finally, current models of miRNA function suggest that miRNAs expressed in a given cell type target transcripts that are already expressed at low levels but avoid housekeeping genes or genes that are expressed in these cells at high levels. These targets may escape detection by microarray analysis. Nevertheless, among transcripts regulated by the Argonaute proteins several were found that are expressed at relatively high levels, suggesting that miRNAs not only silence the expression of undesirable, low-abundance transcripts but may also play a role in fine-tuning the expression of abundant mRNAs (Rehwinkel, 2006).
AGO1 and AGO2 are thought to have nonoverlapping functions in Drosophila. This study shows that these proteins regulate the expression levels of a common set of miRNA targets. The observation that Drosha also regulates these transcripts strongly supports the idea that regulation is mediated by miRNAs. In agreement with this, it was observed that AGO2 can associate with endogenous miRNAs, although less efficiently than does AGO1. In this way, AGO2 may also regulate the expression levels of a subset of miRNA targets. Nonetheless, when miRNA function were assayed by overexpressing miRNAs together with luciferase-based mRNA reporters, it was observed that miRNA-mediated translational repression requires AGO1 but not AGO2. It is therefore possible that in this assay the fraction of miRNAs incorporated into AGO2-containing RISC is too small to observe changes in the expression levels of the reporter. Dicer-1 is involved in miRNA biogenesis and is also required for the assembly of RISC complexes, so these observations suggest that Dicer-1 may load AGO2-containing RISCs with miRNAs, at least to some extent (Rehwinkel, 2006).
A partial functional overlap between AGO1 and AGO2 is also suggested by the observation that these proteins regulate the expression of a common set of transposable elements. It remains, however, to be established whether this regulation occurs via similar mechanisms and whether it happens at the transcriptional or posttranscriptional level (Rehwinkel, 2006).
Apart from the common regulated transcripts, transcripts regulated exclusively by AGO2 but not by Drosha or AGO1 have also been identified, suggesting that AGO2 may regulate the expression of these transcripts by an miRNA-independent mechanism that might involve endogenous siRNAs (Rehwinkel, 2006).
The levels of hid and reaper mRNAs (two experimentally validated miRNA targets increase in cells in which the miRNA pathway is impaired. Moreover, by analyzing changes in mRNA levels, additional miRNA targets have been identified and validated in Drosophila. The observation that miRNA targets change levels following inhibition of the miRNA pathway lends further support to the idea that miRNAs can reduce the levels of the targeted transcripts and not just the expression of the translated protein. Along these lines, it has recently been shown that miRNAs can trigger a strong reduction in target levels in C. elegans. Among the 136 core transcripts, 21% are between 1.5- and 2-fold up-regulated, 73% exhibited changes in the 2- to 5-fold range, and 6% were at least 5-fold up-regulated in AGO1-depleted cells. Thus, although changes in transcript levels can be used to validate miRNA targets, the effects can be modest and, as mentioned above, not all targets can be identified using this approach (Rehwinkel, 2006).
In human cells, the Argonaute proteins localize to P-bodies. These are specialized cytoplasmic foci in which the enzymes involved in mRNA degradation in the 5'-to-3' direction colocalize (e.g., the DCP1:DCP2 decapping complex and the 5'-to-3' exonuclease XRN1. In addition, mRNA decay intermediates, miRNA targets, and miRNAs have been observed in P-bodies, suggesting a functional link between P-bodies and RNA silencing pathways. Consistent with this, it has been shown that P-body components play a crucial role in silencing pathways. In particular, the RNA-binding protein GW182 (a P-body component in metazoa) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing in Drosophila cells. Likewise, human GW182 plays a role in silencing mediated by miRNAs and siRNAs. Finally, the C. elegans protein AIN-1, which is related to GW182, is also required for regulation of a subset of miRNA targets. Together with the observation that miRNAs inhibit cap-dependent but not cap-independent translation initiation, these observations suggest a model in which miRNA targets are stored in P-bodies after translation inhibition, where they are maintained in a silenced state by associating with proteins that prevent translation or possibly by removal of the cap structure. Decapping or simply the storage of miRNA targets in P-bodies may make these mRNAs susceptible to degradation, providing a possible explanation for the reduction in mRNA levels. In agreement with this, depletion of a 5'-to-3' exonuclease in C. elegans partially restores the levels of miRNA targets (Rehwinkel, 2006).
Nevertheless, not all authentic miRNA targets change expression levels. Thus, it is possible that the extent of the degradation depends on the number of miRNA binding sites and/or the stability of the miRNA:mRNA duplexes. It is also possible that the rate of mRNA decay triggered by miRNAs for some targets does not exceed the rate of transcription and that thus the steady-state levels of these targets remain unchanged. It would therefore be of interest to determine whether miRNAs generally cause a reduction in the half-life of targeted transcripts (Rehwinkel, 2006).
Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. This study shows that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. These results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development (Singh, 2011).
The human homolog of Bin3, also called BCDIN3 or methylphosphate capping enzyme (MePCE), was shown to methylate the 5' γ-phosphate on 7SK RNA and to stabilize 7SK RNA in cell culture. This study found that Bin3 associates with and stabilizes 7SK RNA in ovaries and embryos. And, as in human cells, Bin3 activity was specific for 7SK RNA and did not affect U3 RNA or another RNA pol III product, U6 RNA, both of which are methylated by distinct mechanisms. It seem likely, therefore, that Drosophila Bin3 has a similar biochemical activity to its human counterpart despite differing in size and sequence outside the AdoMet binding domain and the highly conserved Bin3-homology domain. Prior attempts to demonstrate protein-arginine methyltransferase activity of Bin3 were negative, consistent with Bin3 methylating RNA rather than protein. In Drosophila, there are two other Bin3-like genes, CG11342 and CG1239, but each is more divergent from the human BCDIN3 within the conserved motif architecture (24% and 39% identity, respectively) than Bin3. It is possible that CG1239, which is expressed in early embryos, could have partially overlapping functions with Bin3 that might contribute to the incomplete penetrance of the bin3 mutations (Singh, 2011).
Putative Bin3 orthologs containing the two conserved motifs are found in at least 70 eukaryotic organisms ranging from the yeast, Schizosaccharomyces pombe to humans, and including Caenorhabditis elegans, Arabidopsis thaliana, Xenopus laevis, and Danio rerio. It is not known what any of these genes do, with the possible exception of the zebrafish bin3 gene which was shown by morpholino knockdown to be important for anterior development and to display RNA splicing defects. Similar defects were sought in splicing of bicoid, caudal, eIF4E, d4EHP, and a control gene, taf1, known to show alternative splicing. No splicing defects were found using a sensitive qRT-PCR approach. It is possible that the splicing defects in zebrafish result from aberrant 5' capping of non-coding RNAs important for splicing (Singh, 2011).
Mammalian 7SK RNA has been studied extensively, but Drosophila 7SK RNA has only been annotated, and prior to this study has not been characterized. This study shows that 7SK RNA is highly expressed in ovaries and embryos and is regulated by Bin3 in a manner similar to that in humans (by BCDIN3). 7SK RNA can be coimmunoprecipitated with Bin3 and Bicoid and may work as a scaffold in translation repression. This is the first indication that 7SK RNA has a function apart from its role in the regulation of the pTEFb transcription elongation factor. While this study focused on Bicoid-dependent regulation, it is likely that 7SK RNA also functions in transcription elongation in other stages of development. Indeed, it was found that Drosophila 7SK RNA mutants showed larval lethality at later stages of development (Singh, 2011).
Bin3 seems to play no role in Bicoid's gene activation function, but instead is crucial for Bicoid-dependent repression of caudal mRNA. Bin3 seems to stabilize Bicoid at the caudal BRE via a mechanism that involves 7SK RNA. As suggested by genetic interaction data, the Bicoid/Bin3/7SK RNA complex may include Larp1, PABP, and Ago2, and target the eIF4E initiation factor (Singh, 2011).
La-related proteins are not restricted to control of transcription elongation. In C. elegans, a Larp1 homolog was shown to be important for downregulation of translation of mRNAs in the Ras-MAPK pathway and to localize to P-bodies, known sites of mRNA degradation, while in mammalian cells, LARP4B plays a stimulatory role in translation initiation. In Drosophila, it has been shown that Larp1 associates directly with PABP independent of RNA and double mutants show enhanced lethality, suggesting that Larp1 facilitates mRNA translation. It is not surprising, therefore, that genetic interactions were observed between bin3 and larp1, as well as with pAbp in the context of caudal translation regulation. Note that it is though that PABP (and Larp1) plays a negative role in translation initiation, as does PABP in the repression of msl-2 mRNA by Sex-lethal (Singh, 2011).
In human cells, BCDIN3 and LARP7 interact cooperatively with 7SK RNA forming a stable core complex that associates transiently with HEXIMS, hnRNPs and the P-TEFb elongation complex. An emerging theme is that 7SK RNA serves as a scaffold for stable association of protein partners. In fact, there is evidence that 5' γ-methylation of 7SK RNA by BCDIN3 may occur co-transcriptionally, but that the modified RNA remains associated with both BCDIN3 and LARP7, which bind 7SK RNA cooperatively. It is proposed, therefore, that Bin3 and Larp1 are associated with 7SK RNA at the caudal BRE, but that 5'-methylation does not necessarily occur there. Consistent with the idea of cooperative binding to 7SK RNA, it was found that larp1 mutation enhanced the bin3 mutant phenotype (Singh, 2011).
Some of the phenotypes observed for bin3 mutants were also observed in mutants of the microRNA miR-184, including oogenesis defects and a cellularization defect. This was the rationale behind including ago2 in the genetic analysis. However, no effect was found of bin3 mutation on levels of several miRNAs, including miR-184, it was surprising to observe a genetic enhancement (albeit mild) of the bin3 phenotype when combined with an ago2 mutation. Ago2 has been shown to bind eIF4E and interfere with mRNA circularization mediated by PABP. However, this occurs in the context of the miRNA/RISC complex, so whether and how Ago2 participates in Bicoid-Bin3 repression is not clear, but it could potentially involve the 7SK RNA component (Singh, 2011).
Finally, no interaction was detected between bin3 and D4EHP, which encodes a previously identified partner of Bicoid important for repressing caudal translation. D4EHP interacts with Bicoid and is thought to directly bind the m7G cap of caudal mRNA, thereby displacing eIF4E and blocking all subsequent steps of initiation. Perhaps the Bin3 mechanism works redundantly with the D4EHP mechanism or perhaps Bin3 helps recruit D4EHP, and so that mutation of bin3 would preclude binding of D4EHP to the initiation complex. Thus, bin3 mutation would be epistatic to the D4EHPCP53 mutation. Further investigation will be needed to determine relationship between these two pathways (Singh, 2011).
Bin3 is unlikely to be a dedicated Bicoid interactor and probably has roles as an RNA methyltranferase in many distinct pathways throughout development. In adults, quantitative trait transcript analysis linked bin3 with sleep-wake cycling. While studying Bin3's role in embryonic patterning, strong oogenesis defects were observed, particularly in bin3 null mothers, although other allelic combinations also revealed similar defects, especially at 29°C. Specifically, bin3 loss-of-function mutants showed dorsalized egg shell phenotypes. Conversely, bin3 overepressing lines showed strong ventralized egg shell patterns that appear to result from a failure of the dorsal appendage primordium to resolve into two domains along dorsal midline. These defects are similar to those of early D-V patterning mutations in the grk pathway, and probably do not result from defects that occur in later during morphogenesis step (Singh, 2011).
bin3 loss-of-function mutants resembled mutations in capicua, squid, cup and fs(K10), among others, while bin3 overexpressing lines resembled grk and pAbp mutations. Interestingly, mechanisms for translation repression of unlocalized grk mRNA feature prominently in the D-V patterning pathway, with squid and cup playing a critical role in repression via interaction with eIF4E, and PABP55 being important for release of that repression. Staining of bin3 mutant ovaries revealed a delocalized signal for Gurken protein but not for grk mRNA. Given the role of Bin3 in translation regulation, and the egg shell phenotypes of bin3 mutations, it seems plausible that Bin3 plays a role in negative regulation of grk translation (Singh, 2011).
Results presented in this study show that Bin3 plays a critical role during both oogenesis and embryonic development. In embryos, Bin3 is required for Bicoid to establish the Caudal protein gradient. Bin3 binds 7SK RNA and likely works by methylating 7SK RNA and stabilizing a repressive complex that assembles on the Bicoid-response element in the 3' UTR of caudal mRNA. Bin3's role during oogenesis is less clear, but based on the observed eggshell phenotypes in bin3 mutants, and gurken expression, Bin3 could play a similar role to help ensure that grk mRNA is translated only in the anterior-dorsal region of the oocyte (Singh, 2011).
A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq was performed in two different embryonic cell lines; AGO2 was found to localize to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome (Moshkovich, 2011).
This study provides the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is dependent on CTCF and CP190 binding and may be partially achieved via looping interactions among cis-regulatory regions and promoters. It is proposed that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Moshkovich, 2011).
These results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 on polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in nonrepetitive euchromatic but not repeat-rich regions, as determined by genome-wide ChIP-seq. It is suggested that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the post-transcriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function (Moshkovich, 2011).
Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2. Second, AGO2, but not Dcr-2, is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity (Moshkovich, 2011).
An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. CP190 mutants were found to remain competent for silencing, suggesting that AGO2 chromatin association is not required for RNAi. Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated (Moshkovich, 2011).
A unique positive role for AGO2 but not other RNA silencing factors was identified in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wild-type levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis (Moshkovich, 2011).
This study shows that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, it was found that the Abd-B RB promoter interacts frequently with Fab-7, Fab-8, and the iab-8 enhancer and, moreover, that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high-frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus (Moshkovich, 2011).
AGO2 is recruited to chromatin insulator sites as well as noninsulator sites in a CTCF/CP190-dependent manner. It is speculated that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions. Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190-dependent looping interactions (Moshkovich, 2011).
AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities. However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis-regulatory elements that can maintain looping interactions of this locus, suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body (Moshkovich, 2011).
AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2. This study found that the AGO251B-null mutation has no effect on Fab-X PRE pairing-dependent silencing on sd as assayed in that study, and genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements that long-range interactions are dependent on insulator sequences and not PREs. Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures (Moshkovich, 2011).
It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In Caenorhabditis elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA. Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation. Recently, it has been shown that Schizosaccharomyces pombe Ago1 participates in surveillance mechanisms to prevent readthrough transcription of mRNA. However, the majority of Ago1 associates with heterochromatic regions, and it is not clear thus far whether Ago1 directly associates with euchromatin or acts post-transcriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms (Moshkovich, 2011).
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