Dicer mRNA is expressed in embryos, in S2 cells and in adult flies, which is consistent with the presence of functional RNAi machinery in all of these contexts (Bernstein, 2001).
A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).
The dissociation of Maelstrom from nuage particles in aubergine and spn-E backgrounds was intriguing in light of their requirement in RNAi in Drosophila spermatogenesis and late oogenesis. Importantly, proteins (or homologs) of RNAi pathway components also act in micro RNA (miRNA) processing. Since miRNAs have been shown to regulate RNA translation, it is conceivable that miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage could represent a step in the generation of specificity in translational control in the germline. To explore this potential relationship between nuage and RNAi/miRNA processing pathways, the localization of additional RNAi components was examined in wild-type and maelstrom ovaries. Argonaute1 and Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila. Dicer is the core RNase of RNAi in Drosophila; it is also required for production of the small RNA effectors of the RNAi and miRNA pathways in C. elegans. In vertebrate cell lines, Dicer is primarily cytoplasmic. In wild-type Drosophila ovarioles, Dicer and AGO1 appear uniform and cytoplasmic in nurse cell cytoplasm; AGO2 appears cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1 distribution is relatively unperturbed. However, AGO2 and Dicer are both dramatically mislocalized in maelstrom ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often perinuclear foci in nurse cells. AGO2 is observed in perinuclear regions of nurse cells, which, by contrast, can colocalize with Vasa in nuage (Findley, 2003).
To determine whether the Dicer enzyme is involved in RNAi in vivo, Dicer activity was depleted from S2 cells and the effect on dsRNA-induced gene silencing was tested. Transfection of S2 cells with a mixture of dsRNAs homologous to the two Drosophila Dicer genes (Dicer-1 and CG6493/Dicer-2) resulted in a roughly 6-7-fold reduction of Dicer activity either in whole-cell lysates or in Dicer immuno-precipitates. Transfection with a control dsRNA had no effect. Qualitatively similar results were seen if Dicer mRNA was examined by Northern blotting. Depletion of Dicer substantially compromises the ability of cells to silence an exogenous, green fluorescent protein transgene by RNAi. These results indicate that Dicer may be involved in RNAi in vivo. The lack of complete inhibition of silencing may result from an incomplete suppression of Dicer or may indicate that in vivo guide RNAs may be produced by more than one mechanism (Bernstein, 2001).
RNA silencing phenomena, either the regulation of mRNA translation or regulation of mRNA degradation, intersect at the ribonuclease Dicer. In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. siRNAs and miRNAs then direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). Mutations have been characterized in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of micro RNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 (but not Dicer-2) is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila (Lee, 2004).
An important though not absolute role has been established for Dcr-2 in Drosophila RNAi. Since another Dicer (Dcr-1) is present in Drosophila, it is possible that it has a redundant function with Dcr-2. The dcr-1 gene is located at 94C4 on the third chromosome, and a mutation was found that mapped by linkage and complementation analysis to that region. The coding sequence of dcr-1 in the mutant contained a premature stop codon such that the truncated product lacks the PAZ and RNase III domains. Moreover, dcr-1 mRNA is not detectable in the mutant as determined by RT-PCR and Northern blot analysis, suggesting that the transcript is unstable when truncated Dcr-1 protein is produced. This transcript null dcr-1Q1147X mutant exhibited an eye color phenotype when assayed in a GMR-wIR genetic background. The mutant appeared to partially suppress silencing by GMR-wIR, with patches of dark orange eye color. In addition, the eye was half its normal size, the organization of ommatidial facets was disrupted, and sensory bristles were missing over the eye surface. Other bristles, which flank the eye surface, were sometimes absent or exhibited hyperplasia (Lee, 2004).
Despite an effect on white gene silencing, the dcr-1 mutant had normal levels of wIR siRNAs. This observation is consistent with Dcr-2 processing the great majority of wIR dsRNA. It is further consistent with a central role for Dicer helicase activity in dsRNA processing, since Dcr-1 lacks a DExH-box helicase domain. If the dcr-1 mutant has normal dsRNA processing, why is it partially disrupted for gene silencing? To answer this, clones of homozygous dcr-1 mutant germ cells were generated in heterozygous females, and then dcr-1 mutant eggs were injected with either dsRNA or siRNA complementary to bicoid transcripts. Loss of bicoid mRNA was measured as a consequence. dcr-1 mutant eggs exhibit an impaired RNAi response to dsRNA and siRNA. Six-fold more bicoid mRNA was present in dcr-1 mutant eggs compared to wild-type eggs after either dsRNA or siRNA treatment. This result indicates that Dcr-1 acts downstream of siRNA production in the RNAi pathway. Dcr-1 plays an important though not absolute role in siRNA-dependent RNAi. Since Dcr-2 is also required downstream of siRNAs, these data suggest that Dcr-1 and Dcr-2 function might be partially redundant in some downstream activity (Lee, 2004).
Dcr-1 is essential to generate mature miRNAs. This role was demonstrated by analyzing miRNA levels in dcr-1 mutant eggs. No mature miRNAs belonging to the miR-2 group were detected in dcr-1 mutant eggs. Thus, Dcr-1 is critical for miRNA production whereas Dcr-2 is required primarily for siRNA production (Lee, 2004).
Although Dcr-1 and Dcr-2 preferentially produce different types of small RNAs, both are required for efficient siRNA-dependent mRNA degradation. Does this dual requirement extend to the miRNA pathway as well? To test this possibility, a genetic assay for miRNA-dependent gene silencing was used in dcr-1 and dcr-2 mutants. Several classes of motifs are present in the 3'UTR regions of the E(spl) and Bearded genes. The 3'UTR motifs are complementary to a variety of miRNAs, and they mediate posttranscriptional repression of gene expression. A series of reporter transgenes was constructed that mimics this posttranscriptional repression. The reporter genes contain a constitutive promoter from armadillo, lacZ coding sequence, and the 3'UTR from the Bearded or E(spl)m8 gene. When the reporter contains a wild-type Bearded 3'UTR, its expression in the developing eye disc is very weak. It is somewhat more strongly expressed in the eye disc posterior to the morphogenetic furrow and is equally weak in the anterior eye disc and antennal disc. When the reporter contains a Bearded 3'UTR with its three B motifs mutated, expression is ubiquitously strong in the eye and antennal discs, confirming that the B motifs mediate a silencing effect on gene expression (Lee, 2004).
Expression of a wild-type reporter gene was examined in clones of mutant dcr-2 cells that were generated in the developing eye disc. Clones expressed the reporter at levels indistinguishable from wild-type tissue, indicating that Dcr-2 is not required for this gene silencing mechanism. In contrast, expression of a wild-type reporter gene in clones of mutant dcr-1 cells was much stronger than in wild-type tissue. The derepressive effect of the dcr-1 mutation requires intact B motifs in the Bearded 3'UTR, since mutant clones did not affect expression of a reporter gene with mutated B motifs. These results argue that dcr-1 but not dcr-2 is necessary for posttranscriptional gene silencing that is mediated by a miRNA mechanism. This conclusion is also validated by other mutant phenotypes associated with each gene. Loss of dcr-1 has profound effects on Drosophila development within both somatic- and germ-lineages, whereas loss of dcr-2 appears to have little or no effect on development (Lee, 2004).
The dcr-1 mutant clones exhibited an interesting pattern of reporter expression. Clones in the antennal disc and eye disc, anterior to the morphogenetic furrow, exhibited little or no derepression of the reporter gene. Typically, only a few mutant cells in each clone had high levels of reporter gene expression. No overt cell differentiation occurs in this region of the eye disc. In contrast, almost all eye disc clones posterior to the furrow, where cell differentiation actively occurs, exhibited extensive derepression. Many, if not most, mutant cells in a clone exhibited this behavior. A boundary of reporter gene expression within a clone could be detected if the clone was bisected by the furrow. This boundary coincided with the morphogenetic furrow. Three interpretations seem possible. (1) Different sets of miRNAs repress the reporter in different regions of the eye disc, one set of which requires Dcr-1 and one set of which does not. However, all of these miRNAs would have to act through the 3'UTR binding sites, since a mutated reporter is constitutively derepressed. Thus, this interpretation is not favored. (2) Dcr-1 might not be essential in anterior disc cells because Dcr-2 or another factor substitutes if Dcr-1 is missing. (3) Anterior disc cells may contain miRNAs that were originally generated in dcr-1+ progenitor cells, and may therefore not require dcr-1. Passage of the morphogenetic furrow may trigger miRNA turnover, resulting in renewed dependence on dcr-1 posterior to the furrow (Lee, 2004).
The RNAi pathway can be divided into discrete biochemical steps: dsRNA processing, maintenance of siRNA 5' phosphate termini, siRNA loading into siRISC, and siRISC-based cleavage of target mRNA. It was confirmed that Dcr-2 but not Dcr-1 is required for dsRNA processing by incubating radiolabeled dsRNA substrate in lysates made from mutant embryos and monitoring siRNA formation. Labeled 21-23 nucleotide RNAs were readily detectable from reactions with wild-type and dcr-1 lysates, but were greatly reduced in reactions with dcr-2 lysate (Lee, 2004).
The genetic experiments suggested a role for Dcr-1 and Dcr-2 downstream of dsRNA processing. Therefore, lysates made from mutant embryos were assayed for steps downstream of siRNA production. siRNAs require 5' phosphate termini for proper association with RISC, and the 5' phosphates are maintained by a kinase that recognizes siRNAs (Nykanen, 2001). Both wild-type and dcr-2 mutant lysates efficiently converted synthetic siRNAs bearing 5' hydroxyl groups into 5'-phosphorylated forms, indicating that the mutant lysates have normal end-maintenance activity (Lee, 2004).
Nykanen (2001) reported that siRNAs are incorporated into a 350 kDa complex when incubated with embryo lysate. The complex is converted to active siRISC using ATP hydrolysis. Radiolabeled siRNA was incubated with wild-type or dcr-2 mutant embryo lysates in the presence of ATP, and then the products were size-fractionated by gel filtration chromatography. siRNA was predominantly associated with a 350 kDa complex in wild-type lysate. However, most of the siRNA in the dcr-2 mutant lysate fractionated as if unbound by proteins, indicating that Dcr-2 is essential for siRNA entry into functional complexes. Thus, dcr-2 lysates should be defective for siRNA-directed mRNA cleavage. SiRNA complementary to a radiolabeled target mRNA was coincubated with embryo lysate. Incubation in wild-type lysate produced a truncated mRNA whose length was consistent with it being the 5' cleavage product. Incubation in dcr-2 lysate generated 5- to 15-fold less cleavage product. This indicates that Dcr-2 is required for mRNA target cleavage by a siRNA, and is consistent with the in vivo observations (Lee, 2004).
To examine siRISC formation in dcr-1 mutant lysate, native gel electrophoresis was used. Gel filtration chromatography requires large lysate volumes, which could not be obtained from dcr-1 embryos. Pham (2004) has developed a gel electrophoretic method to characterize siRNA complexes. The R1 gel complex corresponds to Dcr-2 and R2D2 proteins bound to labeled siRNA. R2 complex appears to be an intermediate that links R1 to a third complex, R3. The R3 complex corresponds to a siRISC that is competent to cleave cognate mRNA (Pham, 2004). To address the role of Dcr-1 in complex formation, complexes were sought in a dcr-1 mutant lysate. R1 complex was detected, but its mobility was slightly shifted and more heterogeneous. No complex with comparable mobility to R2 was detected. The data indicate that proper formation of the R2 intermediate from the R1 precursor complex is dependent upon Dcr-1 (Lee, 2004).
Bacterial RNase III is an antiparallel dimer containing a deep cleft within the catalytic domain. At each end of the cleft lies a symmetric cluster of acidic residues that are conserved among RNase III enzymes. Some of the residues at each end coordinate a divalent metal ion (Mg2+) that is essential for the nucleophilic attack on the RNA phosphodiester bonds at each active site. The metal ion makes a bidentate interaction (inner-sphere and outer-sphere) with an invariant glutamate residue. In one model of catalysis, the nucleophile is used twice, thereby cleaving both strands. In another model, based on a crystal structure, each active site uses two separate clusters of residues to cleave the two phosphodiesters of the RNA helix. One cluster involves the metal ion, and the other cluster acts independent of the metal ion (Lee, 2004 and references therein).
Dicers contain two catalytic domains, which based on the structure of bacterial RNase III, might fold into a pseudodimer structure or might associate within a dimeric holoenzyme. In either scenario, the catalytic repeats contain many of the invariant acidic residues implicated in RNase III catalysis. Moreover, like bacterial RNase III, Dicers cleave dsRNA to produce fragments with 3' overhangs, and with 5'-phosphate and 3'-hydroxy termini in a reaction that requires a divalent metal ion. On this basis, the chemistry of phosphodiester hydrolysis is likely to be similar. Accordingly, certain invariant residues were mutated within Dcr-2 that ware predicted to specifically disrupt phosphodiester hydrolysis. E1371 and E1617 in the first and second RNase III repeats, respectively, are homologous to the E residue that extensively interacts with Mg2+ in the bacterial holoenzyme. An E->K mutant in E. coli RNase III fails to cleave dsRNA, but still binds the dsRNA substrate. To elucidate the functions of the homologous residues in Dcr-2, single and double E1371K and E1617K substitution mutants of dcr-2 were generated and the mutant genes were transformed into a Drosophila strain null for dcr-2. Their ability to silence white expression in a GMR-wIR background was tested. The single mutants gave barely detectable silencing activity, while the double mutant gave no detectable silencing. Thus, E1371 and E1617 are essential for Dcr-2 activity in vivo (Lee, 2004).
Two other conserved acidic residues in bacterial RNase III form an interdomain bridge near each metal binding site, but do not coordinate the metal. Interestingly, all known Dicers have acidic residues in homologous positions of the first domain repeat, but have nonacidic residues in the homologous positions of the second domain repeat. To test the functionality of the residues in the first domain, an E1210V or E1237A substitution was introduced into the dcr-2 gene. The same substitutions at the homologous positions of E. coli RNase III abolish activity. However, both dcr-2 point mutants fully rescued the dcr-2 null phenotype, indicating that E1210 and E1237 are not critical for Dcr-2 activity. These results are consistent with the notion that the nonacidic partner residues in the second repeat normally render these clusters nonfunctional (Lee, 2004).
These genetic and biochemical analyses support the idea that siRISC activity is dependent on Dcr-1 and Dcr-2. Pham (2004) has observed both Dcr-1 and Dcr-2 proteins in siRISC that is competent for target cleavage. At least three functions that Dicer could play in siRISC can be imagined. (1) Dicer may stably associate with siRISC after having passed an siRNA molecule to other RISC factors, but has no further role in siRISC activity. This is unlikely since siRNAs can UV-crosslink to Dcr-1 and Dcr-2 in assembled siRISC. (2) Dicer may use its dsRNA binding activity to retain double-stranded siRNA or a siRNA/mRNA duplex within siRISC. (3) The RNase III domain of Dicer may be responsible for RNA cleavage by siRISC. To test this latter possibility, the E1371K and E1617K variants of Dcr-2 were examined for siRISC activity (Lee, 2004).
Lysates were prepared from mutant embryos in which dcr-2E1371K, dcr-2E1617K, or dcr-2E1371K E1617K genes were expressed in place of the endogenous dcr-2 gene. Lysates were incubated with siRNA duplexes and a labeled mRNA substrate, and siRNA-directed cleavage of the substrate was monitored by 5' product formation. All three mutants exhibited normal mRNA cleavage activity in vitro. To demonstrate that the mutant proteins are nevertheless defective for RNase III activity, they were tested for dsRNA processing. Neither dcr-2E1371K nor dcr-2E1617K lysates were able to support dsRNA cleavage to form siRNAs. These data indicate that siRISC activity is unaffected when Dcr-2 RNase III activity is specifically impaired (Lee, 2004).
AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation (Jing, 2005).
The ARE motif (AUUUA) is the most studied cis-acting element responsible for rapid turnover of unstable mRNAs in mammalian cells. In the quest for a genetic system that allows a comprehensive search for components involved in ARE-mediated decay of mRNA, Drosophila S2 cells were examined and it was found that the decay of ARE-containing RNA in S2 cells is regulated in a manner similar to that in mammalian cells. Inhibition of gene expression by RNAi is much easier and more cost effectively conducted in Drosophila S2 cells compared to mammalian cells: this allowed for an investigation of a large number of genes for their involvement in ARE-mediated RNA decay. Surprisingly, knockdown of Drosophila Dicer1 gene expression leads to stabilizing an ARE-RNA reporter. Further studies revealed that Drosophila Ago1 and Ago2 are required for ARE-mediated RNA degradation, suggesting involvement of the miRNA system. It was then confirmed that human Dicer is required in ARE-RNA degradation in HeLa cells, which implies that this underlying mechanism is conserved in the mammalian cells. Given the involvement of Dicer in HeLa cells, it was reasoned that miRNA(s) are involved in ARE-mediated RNA decay and a search was conducted for miRNAs that possess a complementary sequence to the canonical AUUUA sequence of ARE. miR16 is a potential candidate due to the presence of the sequence UAAAUAUU, and it was shown that downmodulation and overexpression of miR16 increases or decreases, respectively, the stability of a RNA reporter containing ARE of TNF or Cox2, but not uPAR. Furthermore, it was determined that the regulation of ARETNF-RNA decay by miR16 is sequence specific. Just as with Dicer, a function of Ago family members in ARETNF-RNA degradation is likely to be the processing of miR16. However, the interaction with the ARE binding protein TTP indicates that Ago/eiF2C family members also play a crucial role in the targeting of miR16 to ARE. These data demonstrate the involvement of miR16 in controlling ARE-RNA turnover and suggest that cooperation of miRNA and ARE binding proteins is essential in the recognition of ARE and in triggering mRNA degradation (Jing, 2005).
Studies have shown that the ability of miRNA to target mRNA is directed by the pairing of miRNA to mRNA. The ARE-complementary sequence in miR16 is indeed required for miR16 function in destabilizing ARE-RNA. However, pairing with no more than an eight-base ARE-sequence may not be sufficient for miR16 to target ARE-RNA. In addition, the pairing of miR16 to ARE is not in the 5′ region of miRNA, which is believed to be more critical in causing gene repression than the 3′ region. It is speculated, then, that TTP is a factor that assists miR16 targeting to ARE sequences due to its ability to interact with the ARE and RISC complex. This explains why miR16-mediated ARE-RNA instability requires TTP. In addition, the requirement of miR16 in TTP-mediated destabilization of ARE-RNA suggests that targeting of miR16 to ARE is a necessary step for RNA degradation (Jing, 2005).
ARE sequences from different mRNA can vary dramatically, with some containing multiple AU-rich elements that allow for simultaneous interaction with more than one miRNA. This could influence the ability of miRNA to promote RNA degradation because of the potential synergistic effect of miR16 to bind to multiple sites. This synergism has been demonstrated in a study that shows the addition of multiple binding sites of CXCR4 siRNA into 3′UTR of a reporter results in more translation inhibition than expected when summing up the individual effects of each binding site. The number of pairs that miR16 can form with different ARE sequences varies from five to eight, and the strength of interaction between miR16 and different AREs in a given mRNA may also vary. The number of miRNAs targeted to an mRNA and the strength of the interaction may both contribute to the quantitative control of mRNA turnover or translation. Perhaps since no more than six pairs can form between miR16 and ARE of uPAR and since uPAR has only one AUUUA motif in the 3′UTR, miR16 does not have a significant effect on the stability of mRNA containing uPAR 3′UTR (Jing, 2005).
miR16 is conserved in mammals. Although a homolog of miR16 has not been found in Drosophila, miR289 contains UAAAUAUUUA, and four other known Drosophila miRNAs contain a UAAAU sequence. Among them, at least miR277, miR289, and miR304 are expressed in S2 cells. 2′-O-methyl oligonucleotides were used to test for Drosophila miRNA that could be involved in ARE-RNA degradation in S2 cells. The anti-miR289 oligo significantly stabilizes mRNA containing TNF-α ARE, while the other four oligos have no or very modest effects on the stability of ARETNF-RNA. miR289 has a similar effect on the stability of AREIL-6-RNA and AREIL-8-RNA. Sequence comparisons showed that miR289 partially complements with ARE, but not the other regions of these 3′UTRs. Thus, miR289 is likely to be a miRNA that has a role in regulating ARE-RNA in S2 cells (Jing, 2005).
Though the association of miR16 with ARE-RNA in the presence of TTP and S-100 in vitro has been demonstrated, the exact mechanism of miRNA targeting of ARE and regulation of RNA degradation remains undetermined. Because of the similarity between siRNA and miRNA in regulating gene expression, miR16-mediated ARE-RNA degradation could be similar to siRNA-mediated mRNA decay. It is theoretically possible that the targeting of ARE with miRNA leads to mRNA cleavage at the targeting site since RISC has been shown to be an RNA endonuclease in vitro. However, translational suppression caused by miRNA or imperfect pairing of siRNA suggests that endonuclease activity is not always associated with RISC. Since ARE-RNA degradation is believed to be initiated by deadenylation and subsequent targeting by the exosome pathway, and since endocleaved ARE-RNA was not detected in the experimental system that was used, it is believed that the RISC involved in ARE-RNA decay is not associated with endonuclease activity. At the present, it is not clear if RISC can execute an exonuclease function, although an exonuclease, Tudor-SN, has been found in the RISC complex. TTP has been shown to bind to extended ARE sequences by virtue of its zinc finger and associates with components of exosomes; this study shows that TTP is also associated with eiF2C/Ago family members. A recent study reported that an exosome associated DexH box helicase facilitates ARE-RNA deadenylation and decay in mammalian cells. Interestingly, a C. elegens homolog of this DexH box protein has been shown to interact with a protein complex containing Dicer, RDE-1, and RDE-4. It appears that ARE binding proteins, miRNA, deadenylase, and exosomes cooperate with each other in regulating mRNA degradation. A model is favored in which TTP binds to an ARE and transiently interacts with the RISCs that scan mRNA. When a RISC containing miR16 encounters TTP, it stays with ARE and TTP due to base complementarity between miR16 and ARE. It is conceivable that RISC, in conjunction with TTP, serves to recruit proteins for deadenylation and/or exosomes for mRNA degradation (Jing, 2005).
Hundreds of miRNAs have been identified, but the targets of most miRNAs are unknown. Since perfectly or nearly perfectly paired sequences can only be found for a few miRNAs, computational as well as experimental approaches have been developed to identify potential miRNA targets that do not contain perfect complementary sequences. Although these approaches have been shown to be very useful, ARE was not identified as the target of miR16 through currently available computer programs. The current data suggest that additional factors, such as sequence-specific RNA binding proteins, needs to be considered in studying the function of miRNA. As in the case of miR16, many miRNAs may require specific proteins in binding to their mRNA targets. The role of many miRNAs may need to be studied, not only in the context of miRNA-mRNA interaction, but also the interaction of miRNA complexes with other proteins (Jing, 2005).
One of the key characteristics of stem cells is their capacity to divide for extended periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. This study reports the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, has revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, it is concluded that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals which normally stop the cell cycle at the G1/S transition (Hatfield, 2005).
MicroRNAs and short interfering RNAs (siRNAs), processed by the type III double-stranded RNase Dicer, function in an RNA-based mechanism of gene silencing. Most characterized miRNAs from animals repress gene expression by blocking the translation of complementary messenger RNAs into protein; they interact with their targets by imperfect base-pairing to mRNA sequences within the 3' untranslated region (3' UTR). Experimental evidence has suggested that small RNAs regulate stem cell character in plants and animals. Moreover, some miRNAs are differentially expressed in stem cells, suggesting a specialized role in stem cell regulation. However, the molecular mechanisms underlying stem cell control by miRNAs are not understood (Hatfield, 2005).
To determine the role of miRNAs in the control of stem cell biology, processing of all miRNAs in stem cells was specifically eliminated. The Drosophila genome contains two Dicer isozymes: Dicer-1 and Dicer-2. Dicer-1 (Dcr-1) is essential for processing miRNAs, whereas Dicer-2 (Dcr-2) is required for siRNAs; loss of Dcr-1 completely disrupts the miRNA pathway and only has a weak effect on the siRNA pathway. Using Drosophila GSCs as a model system, Dcr-1 activity was impaired with two dcr-1 alleles: dcr-1 d102 and a null dcr-1 Q1147X. Drosophila oogenesis depends on the presence of self-renewing GSCs in the adult ovary, as has recently been reported in a mammalian system. The continuous division of GSCs generates an array of progressively developed egg chambers in wild-type ovarioles (Hatfield, 2005).
Analysis of dcr-1 mutant clones in the Drosophila ovary 12 days after clone induction has revealed a marked depletion of developing egg chambers. In contrast, dcr-2 null mutant GSCs produced a normal progression of egg chambers. These data suggest that Dcr-1 is required for efficient germline production. Although dcr-1 mutants show reduced numbers of gametes, most developing gametes appear morphologically normal (although they exhibit polarity defects. Therefore potential problems in GSC maintenance, identity and division were analyzed. Clonal experiments revealed that the percentage of germaria with clonal stem cells at different time points after clone induction was similar in the dcr-1 Q1147X mutant and the wild-type control, suggesting that the loss of cysts in dcr-1 mutants is not primarily due to problems in the maintenance of GSCs (Hatfield, 2005).
To determine whether reduced cyst production in dcr-1 germaria is due to altered GSC fate, the identity of the dcr-1 mutant GSCs were analyzed. Female GSCs are identified by their location and the expression patterns of three markers: the presence of Adducin, a protein present in the spectrosome; the presence of phosphorylated Mad protein (P-Mad), indicating TGF-beta, and the absence of Bam, repressed by the TGF-beta. Thedcr-1 Q1147X GSCs showed normal spectrosome morphology and position, and normal TGF-beta pathway activity. Furthermore, as with wild-type GSCs, dcr-1 Q1147X GSCs did not stain positively for the Bam protein. From these analyses, it is concluded that decreased cyst production from dcr-1 Q1147X GSCs does not result from either a loss of GSCs or a change in their identity (Hatfield, 2005).
The frequency of cell division in dcr-1 Q1147X GSCs was impaired. Examination of individual germaria containing a single heterozygous GSC and a single dcr-1 Q1147X mutant GSC revealed that GSCs lacking Dcr-1 activity produced cysts at a frequency that was reduced to 18% of normal levels (41% for dcr-1 d102). In contrast, the frequency of division was not significantly reduced for GSCs that were homozygous for the dcr-2 mutation or for the isogenized parental chromosome from which the dcr-1 mutant alleles were generated. Thus, Dcr-1 is required cell autonomously in GSCs for the cell divisions that produce developing cystoblasts (no obvious defect in cyst division was observed (Hatfield, 2005).
To determine whether the reduced cyst formation reflected a block in the normal cell cycle programme, the distribution of cell cycle stages was analyzed in mutant dcr-1 Q1147X GSCs by staining mosaic germaria with antibodies against different cell cycle markers. An increase in the number of dcr-1 mutant GSCs staining positive for Cyclin E (CycE) was observed using two independent dcr-1 alleles. In contrast, GSCs that are homozygous for dcr-2 or the parental chromosome express CycE with frequencies similar to those of wild-type GSCs. Furthermore, pulse labelling of ovaries with the nucleotide analogue 5-bromodeoxyuridine (BrdU) revealed that the number of dcr-1 Q1147X mutant GSCs in S phase was reduced. Similarly, the number of dcr-1 Q1147X mutant GSCs staining positive for Cyclin A (CycA), Cyclin B (CycB) and the mitotic marker Phosphohistone-3 (PH3) was reduced. These data indicate that perturbation of the miRNA pathway by mutant dcr-1 in GSCs delays the cell cycle at the G1/S transition (Hatfield, 2005).
Whether loss of Dcr-1 function has similar consequences on the cell cycle in the GSCs of male flies was tested. Each male testis contains approximately ten GSCs surrounding a somatic structure called the hub. Similar to female GSCs, the number of male GSCs staining positive for CycE was increased in dcr-1 mutants. These data show that Dcr-1 also functions in the male GSC niche, and suggest that Dcr-1 has a conserved role in GSC division (Hatfield, 2005).
To test the possibility that the miRNA pathway might be a general cell cycle regulator, other cell types were tested to determine whether the G1/S delay and reduced cell division frequency would also be observed in other mitotically dividing dcr-1 mutant cells. dcr-1 Q1147X clones in imaginal discs revealed that the number of CycE-positive cells was not increased in mutant cells. The number of dcr-1 Q1147X mutant cells in imaginal discs was approximately equal to the number of marked wild-type cells that descended from a common parent cell, indicating that the frequency of cell division in imaginal disc cells is not reduced in a dcr-1 mutant. dcr-1 Q1147X dividing germline cysts express CycE at a frequency comparable to that of wild-type dividing cysts, suggesting that the mitotic cystoblast cell divisions are not affected in dcr-1 mutants. Therefore, the reduction in cell division frequency observed in the dcr-1 mutant germ line is specific to the GSC division. Together, these data suggest that the miRNA pathway has a specific role in regulating stem cell division (Hatfield, 2005).
The potential cause for the G1/S arrest was explored by examining the expression of Dacapo (Dap; a homologue of the p21/p27 family of cyclin-dependent kinase (CDK) inhibitors) in dcr-1 Q1147X mutant GSCs. The transition between the G1 and S phases of the cell cycle is negatively regulated by Dap. Dap protein traps the CycE/CDK2 complex in a stable but inactive form, and elevated levels of Dap lead to cell cycle arrest at the G1/S phase transition with prolonged expression of CycE protein. Notably, the number of Dap-positive GSCs increased in the dcr-1 mutant GSC population (Hatfield, 2005).
To determine whether Dap mediates the effect of dcr-1 on the GSC cell cycle, the level of Dap was reduced by 50% in dcr-1 Q1147X mutant GSCs and a partial rescue in cyst production was observed. Furthermore, the number of GSCs staining positive for CycE was reduced to normal levels, demonstrating that the CycE defect observed in dcr-1 mutant GSCs is dependent on Dap. Consistent with this, overexpression of a Dap transgene results in some germaria resembling dcr-1 germline mutants: the germaria are small, containing a few cysts, and had a high number of CycE-positive GSCs. The fact that reduction of Dap levels led to a normal GSC CycE profile, but partial rescue of cyst generation, suggests that Dcr-1 might also regulate later cyst development (Hatfield, 2005).
These data suggest that miRNAs act on stem cell division by reducing the levels of Dap. How is this regulation achieved? It was found that expression of a Dap transgene containing the Dap promoter and essentially all of the endogenous gene except some of the 3' UTR is similar in dcr-1 mutant and wild-type GSCs. These data suggest that the effect of Dcr-1 on Dap regulation in GSCs is at a post-transcriptional level and might involve the 3' UTR region that is missing in the dap-5gm transgene (Hatfield, 2005).
It is proposed that miRNAs are required for GSCs to transit the G1/S checkpoint by repressing directly or indirectly the G1/S inhibitor Dap. Because Dap is a key component of the G1/S transition, it is a plausible target for machinery that assures continuous cell division in a microenvironment in which most of the cells are quiescent. It is proposed that while the TGF-beta pathway -- which can upregulate p21/p27 -- is active in GSCs, miRNAs downregulate Dap to assure the continuous cell division essential for stem cells. This downregulation might be direct, because the Dap 3' UTR contains several predicted miRNA-binding sites. A Dap transgene lacking these sites shows no response to Dcr-1 levels, suggesting that the potential binding sites are responsive to Dicer-1. However, it is also possible that the Dap misregulation in dcr-1 mutant GSCs might be due to a secondary effect of Dcr-1 loss. The finding that miRNAs are required for stem cell division suggests that miRNAs might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle. Because miRNAs are a novel class of genes involved in human tumorigenesis, it is tempting to speculate that miRNAs could have a similar role in cancer cells (Hatfield, 2005).
MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3' untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, the proteome from wild-type oocytes was compared with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3' untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization (Nakahara, 2005).
At the completion of Drosophila oocyte maturation, the number of genes negatively regulated by miRNAs appears to be limited. This study detected 41 of 1,003 proteins that are down-regulated by miRNA production, representing a maximum 4% of genes that might be directly inhibited. Although this value is close to the genome-wide 7% level predicted by sequence comparisons, it is not certain that 4% represents a typical fraction of genes regulated by miRNAs in a cell. First, this estimate reflects only the 1,000 most-abundant proteins at this stage of development. Many less-abundant proteins are not detected by Difference Gel Electrophoresis, and so the fraction of those that are regulated is not known. For example, dcr-1 mutant oocytes exhibit defective translation of Oskar and Gurken proteins, leading to a mild ventralization phenotype. However, these two proteins are too rare to have been detected by the proteomic analysis. Second, it is not known whether the relative fraction of target genes varies during development. Possibly, fewer genes are regulated by miRNAs during oogenesis. It is known that RNAi is activated during oocyte maturation, and that the short interfering RNA pathway depends upon translation of target mRNAs. It is possible that there are fewer miRNA targets in mature oocytes due to dependence on different translation control mechanisms (Nakahara, 2005).
Why are these particular gene products repressed by miRNAs during oocyte maturation? In most animal species, translation serves as the main mechanism to regulate gene expression during oocyte maturation and early embryogenesis. Indeed, oocyte maturation and early embryogenesis proceed without transcription of nuclear RNA, including rRNA. In parallel, no ribosomal proteins are synthesized de novo, and consequently no new ribosomes are produced. In Drosophila, ribosomal protein-mRNA levels are constant throughout oogenesis and embryogenesis, but their translation drops as oogenesis ends and embryogenesis begins. Translation of ribosomal protein-mRNAs then rises in conjunction with the onset of rRNA transcription in the embryo. It was found that the accumulation of ribosomal protein-S2 is inhibited by miRNAs during oocyte maturation, suggesting that the translation block exerted on Drosophila ribosomal proteins is partially mediated by the miRNA pathway. The status of other ribosomal-proteins is not known, because most are basic in charge and would not be detected in the 2D gels used in the analysis (Nakahara, 2005).
A lack of new ribosome production during oocyte maturation and early embryogenesis would also eliminate the need to synthesize other factors involved in protein biogenesis, reflecting a coordinated effort to balance various steps along the biogenesis pathway. This study indicates that the synthesis of several chaperones and other biogenic factors is specifically inhibited during oocyte maturation, possibly for those reasons. Another cellular process that appears to be attenuated by miRNAs at this developmental stage is protein turnover. Why is proteolysis dampened? Possibly, reduced proteolysis allows preexisting ribosomes to remain abundant during the period when they cannot be replenished. Another possibility is that reduced proteolysis links the rate of protein biogenesis to the rate of protein turnover, thereby maintaining steady-state protein levels. Finally, lowered protein turnover during this highly dynamic stage of development would allow for rapid and global accumulation of new proteins necessary for early embryogenesis (Nakahara, 2005).
The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. This study reports the function of Drosophila Piwi, Dicer-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. Piwi is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect Osk or Vasa expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases Osk and Vasa levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, Piwi forms a complex with dFMRP and Dicer-1, but not with Dicer-2, in polar-granule-enriched fractions. Depleting Dicer-1, but not Dicer-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. Because Dicer-1 is required for the miRNA pathway and Dicer-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, the data implicate a crucial role of the Piwi-mediated miRNA pathway in regulating the levels of Osk, Vasa, and possibly other genes involved in germline determination in Drosophila (Megosh, 2006).
It has been nearly a century since the discovery of germ plasm and its function in germline fate determination in diverse organisms. In recent decades, the components and assembly of the polar granule in Drosophila and its equivalent in C. elegans have been effectively explored. Translational regulation has also been implicated in pole plasm for abdominal patterning and germline determination. In addition, germ cell-less (gcl) and mitochondrial large-subunit ribosomal RNAs (mtlr RNAs) have been shown to be required for germline determination. However, the biochemical activities of these molecules remain largely unknown. This study identified Piwi and likely the miRNA machinery as a germ-plasm regulatory activity that is involved in germline fate determination (Megosh, 2006).
Germ-plasm assembly occurs in a stepwise fashion. Step 1 involves the transport of polar granule materials to the posterior end of the oocyte during oogenesis, a process that involves a microtubule-based transport system as well as genes such as cappuccino and staufen. Step 2 is the assembly of polar-granule components at the posterior end, a process that is almost concurrent with the transport and that is completed by stage 12 of oogenesis. A critical component for the assembly is Osk, which determines the pole-cell number in a dose-dependent manner and has the ability to recruit Vasa and Tud as well as to induce pole-cell formation at ectopic sites within the embryo. Three lines of data suggest that Piwi is downstream of Osk, Tud, and Vasa in the assembly process: (1) Osk, Tud, and Vasa appear to assemble normally into the pole plasm in Piwi-depleted developing oocytes; (2) Piwi cannot recruit Osk or Vasa ectopically to the anterior pole, yet Osk can recruit Piwi to the anterior pole; (3) Osk, Tud, and Vasa all have both germline determination and posterior-patterning functions, but Piwi does not appear to have a detectable function in patterning (Megosh, 2006).
Although the assembly of polar-granule components occurs in a hierarchical fashion, there is growing evidence for interactions between polar-granule components beyond what is required for assembly. For example, a regulatory relationship between nanos and tudor has been reported. In nanos mutant embryos, both Tudor levels and the number of pole cells increase. Other experiments suggest that the presence of mtlrRNA in the polar granules is required for stabilization of the polar-granule components Vasa, Gcl, nos mRNA, and pgc mRNA. The regulatory function reported in this study for Piwi toward Osk, Vasa, and Nos further supports the interplay and interdependency among pole-plasm components. A previous study implicates osk as a rate-limiting factor for all aspects of pole-plasm function. The results suggest that Piwi, likely working through the miRNA pathway, is also a limiting factor for germ-cell formation. This function of Piwi is likely achieved via regulation of the levels of Osk, Tud, and Vasa, and possibly that of other polar-granule components, in a dose-dependent fashion (Megosh, 2006).
The regulation of Piwi toward the expression of Osk, Tud, Vasa, and Nos appears to be dispensable; Piwi-deficient oocytes and early embryos do not display detectable defects in their expression of Osk, Tud, Vasa, and Nos. This redundancy is likely due to an overlapping function of Piwi with other proteins involved in the RNAi pathway and/or colocalized in nuage during oogenesis; such proteins might include Maelstrom, Armitage, and Aubergine. Among these proteins, Aubergine, a close homolog of Piwi, is a known polar-granule component in early embryos. It regulates the translation of Osk during oogenesis and is required for both pole-cell formation and posterior patterning during embryogenesis (Megosh, 2006).
It is intriguing that Piwi regulates Osk and Vasa expression yet does not display a posterior-patterning phenotype. This function is different from that of Aubergine, so it is possible that Piwi and Aubergine each have their own regulatory targets in addition to Osk and Vasa. The Piwi targets may be specifically involved in maintaining polar-granule localization and may not be subject to Aubergine regulation, whereas Aubergine targets might be involved in both germline determination and posterior patterning. In support of this possibility, it has recently been shown that the generation of certain rasiRNAs shows varying dependencies on Piwi and Aubergine. The regulation of Piwi toward its specific target genes may be activated during oocyte maturation, similar to the oocyte maturation-dependent activation of RNAi as observed for aubergine and spindle-E. Thus, Piwi is not required for Osk and Vasa localization during oogenesis but is required for maintaining their localization during embryogenesis. An alternative hypothesis is that Piwi, like Aubergine, also regulates patterning genes but that this function is redundant. This hypothesis, however, does not explain the fact that neither ectopic expression nor overexpression of Piwi causes a detectable defect in posterior patterning (Megosh, 2006).
Given the association of Piwi with Dcr-1 and dFMRP, the Piwi-mediated regulation is likely via the miRNA but not the siRNA mechanism, which is Dcr-2-dependent, or the rasiRNA mechanism, which does not depend on either Dcr-1 or Dcr-2. This hypothesis is further supported by the similar phenotypes observed in embryos depleted of Piwi, Dcr-1, and dFMRP but not Dcr-2. It is possible that Piwi might bind to novel small RNAs to achieve this function, given recent findings that mammalian Piwi subfamily proteins bind to Piwi-interacting RNAs (piRNAs). If so, these novel RNAs must function in a Dcr-1-dependent pathway in the cytoplasm given Piwi's localization to the cytoplasm in early pole cells. The function of the Piwi/DCR-1-mediated miRNA or novel small-RNA pathway in germline specification is very similar to that of other germ-cell regulators, such as gcl and mtlr RNAs, in that these genes are required for pole-cell formation but not for abdominal segmentation. However, unlike embryos from the gcl-bcd females, embryos from the piwi-bcd females exhibit no cell-cycle delays in the anterior nuclei and no significant changes in the morphology of anterior nuclei. Furthermore, GCL mediates a transcriptional repression mechanism [72]. Thus, the effect of the Piwi-miRNA mechanism on pole-cell formation may be distinct from the gcl-mediated mechanism (Megosh, 2006).
It is important to note that the Piwi-mediated miRNA pathway positively regulates the expression of Osk and Vasa, in contrast to the known translational repression role of the miRNA pathway. In support of this observation, the Piwi ortholog in the mouse, MIWI, also appears to positively regulate gene expression, likely by enhancing mRNA stability and translation. Alternatively, it is possible that Piwi regulates an unidentified intermediate protein whose function is to repress the expression of Osk and Vasa (Megosh, 2006).
piwi is essential for the self-renewal of adult germline stem cells in Drosophila. Recent studies have demonstrated that the miRNA pathway is involved in division and self-renewal of adult germline stem cells in the Drosophila ovary. This study further connects Piwi and the miRNA pathway and reveals their crucial role in germline fate determination during embryogenesis. These observations suggest that the germline and stem cells may share a common miRNA-mediated mechanism in defining their fates. Given the high degree of conservation of the miRNA machinery during evolution, this pathway may function in diverse organisms in determining the germline and stem cell fates (Megosh, 2006).
MicroRNAs (miRNAs) regulate gene expression by controlling the turnover, translation, or both of specific mRNAs. In Drosophila, Dicer-1 (Dcr-1) is essential for generating mature miRNAs from their corresponding precursors. Because miRNAs are known to modulate developmental events, such as cell fate determination and maintenance in many species, whether a lack of Dcr-1 would affect the maintenance of stem cells (germline stem cells, GSCs; somatic stem cells, SSCs) in the Drosophila ovary was investigated by specifically removing its function from the stem cells. The results show that dcr-1 mutant GSCs cannot be maintained and are lost rapidly from the niche without discernable features of cell death, indicating that Dcr-1 controls GSC self-renewal but not survival. bag of marbles (bam), the gene that encodes an important differentiating factor in the Drosophila germline, however, is not upregulated in dcr-1 mutant GSCs, and its removal does not slow down dcr-1 mutant GSC loss, suggesting that Dcr-1 controls GSC self-renewal by repressing a Bam-independent differentiation pathway. Furthermore, Dcr-1 is also essential for the maintenance of SSCs in the Drosophila ovary. These data suggest that miRNAs produced by Dcr-1 are required for maintaining two types of stem cells in the Drosophila ovary (Zin, 2007).
This study has demonstrated that Dcr-1 is required for the maintenance of GSCs and SSCs in the Drosophila ovary. Because Dcr-1 is an essential component of the miRNA pathway in Drosophila, it is further proposed that miRNAs processed by Dcr-1 are essential for controlling self-renewal of GSCs and SSCs. Consistent with this idea, the Dcr-1 partner, Loqs, has also shown to be required for GSC maintenance. Because Dcr-1 is intrinsically required for controlling GSC self-renewal, Loqs functions intrinsically in GSCs for controlling GSC self-renewal. Without one or more miRNAs generated by Dcr-1, ovarian GSCs and SSCs undergo premature differentiation that leads to the depletion of these stem cells in their corresponding niches. This study has also provided insight into how Dcr-1 controls GSC self-renewal. The miRNA pathway controls GSC self-renewal by repressing a bam-independent differentiation pathway. Furthermore, the miRNA pathway is required for the development and growth of later-differentiated germ cells, although it is dispensable for cyst division and oocyte specification. Similarly, the miRNA pathway is also required for follicle-cell proliferation and growth. To further understand how the miRNA pathway controls GSC and SSC self-renewal, it is essential to identify miRNAs and study their functions in GSC and SSCs. Because Dcr-1 contributes to the maintenance of Drosophila ovarian GSCs and SSCs tested so far, it is tempting to speculate that miRNAs may have a general role in maintaining different types of stem cells. It will be of great interest to test this hypothesis and to elucidate the underlying mechanisms as to how miRNAs contribute to the stem cell self-renewal and proliferation (Zin, 2007).
The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition. This study identified two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendritic mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. These findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system (Berdnik, 2008).
To identify genes that are essential for dendrite targeting in Drosophila olfactory projection neurons (PNs), a MARCM-based mosaic forward genetic screen was performed by using novel piggyBac transposon insertions. The insertions LL03660 and LL06357, integrated in pasha and Dicer-1, respectively, were uncovered. Both alleles are homozygous lethal, likely to be null, and referred to as pasha−/− and Dicer-1−/− mutants throughout this study. The pasha−/− allele is an insertion in the 5'UTR, resulting in undetectable Pasha protein in homozygous mutant neurons. The Dicer-1−/− allele is an insertion in the coding region resulting in a truncated 740 amino acid protein lacking the RNase III, PAZ, and dsRNA binding domains (Berdnik, 2008).
The MARCM technique allows visualization and manipulation of PNs in neuroblast and single-cell clones in an otherwise heterozygous animal. Gal4-GH146 was used to label PNs from three neuroblast lineages, anterodorsal (ad), lateral (l), and ventral (v) PNs. Wild-type (WT) adPNs, lPNs, and vPNs target stereotyped sets of glomeruli in neuroblast clones. pasha−/− PNs show two dendrite morphogenesis defects for all neuroblast clones. First, the dendritic density in most glomeruli is drastically reduced. Second, dendritic branches spill into incorrect glomerular classes. Very similar PN dendritic defects were observed in Dicer-1−/− MARCM clones (Berdnik, 2008).
It was confirmed that the transposon insertions in pasha and Dicer-1 are the cause for the mutant phenotype with two further experiments. First, precise excision of both transposons fully revert PN morphogenesis defects. Second, expression of UAS-pasha-HA or UAS-Dicer-1 transgenes fully rescued pasha or Dicer-1 mutant PN phenotypes, respectively, in MARCM experiments. Because Gal4-GH146 is expressed only in postmitotic neurons, these experiments also demonstrate that Pasha and Dicer-1 act in postmitotic neurons to regulate dendrite morphogenesis (Berdnik, 2008).
As expected, in all rescue experiments, Pasha-HA localizes to the nucleus and Dicer-1 is enriched in the cytoplasm of PNs. Endogenous Pasha protein is found ubiquitously in all cell nuclei in the brain center at 18 hr after puparium formation (APF), when PN dendrites organize the proto-antennal lobe prior to olfactory receptor neuron (ORN) axon entry. Moreover, Pasha is undetectable in pasha−/− adPNs and DL1 single neurons (Berdnik, 2008).
To study dendrite targeting with a better resolution, single-cell MARCM clones were examined. WT DL1 single-cell clones (hereafter referred to as DL1 single neurons) always target a posterior, dorsolateral glomerulus and fill the glomerulus with dendritic branches. In pasha−/− PNs, 17/25 DL1 single neurons show stereotyped mistargeting defects: dendrites innervate DL1 more sparsely and also mistarget to several additional glomeruli (VA7m, VC2, VA6, DL2d, and DL5), all of which are partially innervated. 8/25 DL1 single neurons spill their dendrites medially to adjacent glomeruli, mostly D and DL5. Again, Dicer-1 single mutant neurons exhibit similar PN dendrite mistargeting although to a lower frequency. Similar stereotyped mistargeting pattern as in pasha mutants occur in 19/35 DL1 single neurons mutant for Dicer-1, 7/35 single neurons show medially spilled dendrites and 9/35 target normally. The variation of DL1 phenotypes could be caused by perdurance of WT protein in single-cell mutant clones, which might affect Dicer-1 more than Pasha. The stereotyped DL1 targeting defect was not found in more than 1400 other piggyBac insertions screened, supporting the specificity of the mutant phenotype for the miRNA processing pathway (Berdnik, 2008).
MARCM expression of UAS-Pasha-HA in pasha−/− or UAS-Dicer-1 in Dicer-1−/− DL1 single neurons fully rescued dendrite targeting, as is the case of neuroblast clones. These experiments demonstrate that Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate DL1 dendrite targeting (Berdnik, 2008).
To expand the studies of dendrite targeting to other specific PN classes, Gal4-Mz19 was used to label fewer neurons in neuroblast clones. This Gal4 line labels ~6 adPNs that innervate VA1d and DC3 (posterior to VA1d) in WT. In 21/21 pasha−/− adPNs, VA1d/DC3 is sparsely innervated and dendrites are incorrectly targeted to variable glomeruli such as DA1, VA2, and VM7. 23/25 Dicer-1−/− PNs show similar medial mistargeting phenotypes albeit to a milder extent, innervating less distant glomeruli. Similarly, the dendritic density is reduced and incorrect glomeruli are innervated, as in GH146 MARCM experiments. Gal4-Mz19 is also expressed in ~7 lPNs innervating the dorsolateral DA1 glomuerlus in WT. DA1 PN targeting is much less affected in pasha and Dicer-1 mutants. 4/5 pasha mutant and 7/9 Dicer-1 mutant lPNs target normally to DA1 with WT dendrite densities, whereas 1/5 and 2/9 lPNs exhibit additional partial innervation of the adjacent DL3 glomerulus, respectively. Thus, Pasha and Dicer-1 are not required equally in all PN classes, suggesting that potential miRNAs might selectively regulate the targeting of specific classes of PNs (Berdnik, 2008).
In addition to dendrite mistargeting, axon defects were also observed in pasha and Dicer-1 mutants. WT DL1 axons project into the lateral horn (LH) via the mushroom body calyx (MBC) where they form several collateral branches. After entering the LH, DL1 axons always form one characteristic dorsal branch whereas the main branch terminates at the lateral edge of the LH. In pasha and Dicer-1 mutant DL1 single neurons, axons extend along the normal pathway, form collaterals in the MBC, and always reach the LH. However, more than half of the mutant DL1 axons do not reach the lateral edge but stop within the LH. The dorsal branch in the LH is either absent or reduced in length. Adding one copy of a UAS-pasha-HA transgene in pasha or UAS-Dicer-1 in Dicer-1 mutant DL1 single neurons rescued all axon phenotypes: the main branch fully extends to the lateral edge of the LH and the dorsal branch is indistinguishable from WT. Thus, Pasha and Dicer-1 cell autonomously regulate PN axon-terminal elaboration (Berdnik, 2008).
To determine whether the PN dendrite targeting errors are a result of initial mistargeting or failure to maintain stable synaptic connections later, developmental studies were performed. At 18 hr APF, when ORN axons have not yet entered the proto-antennal lobe, WT adPN, lPN, and vPN dendrites have already occupied a large area of the proto-antennal lobe . DL1 single neurons already target their dendrites in the area of the future DL1 glomerulus. In pasha−/− PNs, dendritic elaboration within the proto-antennal lobe is extremely reduced in all neuroblast or DL1 single-cell clones at 18 hr APF. At 50 hr APF, glomeruli become first visible. In WT adPNs, lPNs, and DL1 single neurons, the same stereotyped innervation patterns as in adults are already evident even though the antennal lobe is smaller in its overall size. Dendrites of pasha−/− PNs are reduced in density and spill into lineage-inappropriate glomeruli. Moreover, stereotyped mistargeting of DL1 single neurons is already evident in 4/4 pasha−/− PNs at 50 hr APF (Berdnik, 2008).
These data, in combination with the observation that pasha mutant PN dendrite phenotypes do not vary in brains of 3- and 10-day-old adults indicate that Pasha regulates dendrite elaboration and correct targeting early during development (Berdnik, 2008).
Dicer functions in small RNA maturation across species. Dicer mutants are defective for both transcript destruction and translational repression, suggesting that Dicer is required for the siRNA (small interfering RNA) and miRNA maturation pathway. However, the Drosophila genome contains two Dicer genes, Dicer-1 and Dicer-2, that share similar protein domains but are different in their functions. Dicer-1 and Dicer-2 are both required for siRNA-dependent mRNA cleavage, with Dicer-2 acting in siRNA processing and Dicer-1 acting downstream of siRNA production. However, Dicer-1, but not Dicer-2, is essential for miRNA-induced silencing during translational repression (Berdnik, 2008).
To test whether the siRNA processing pathway is required for PN targeting, use was made of Dicer-2L811fsX mutants that lack the two RNase III domains essential for dsRNA processing. It was found that Dicer-2L811fsX mutant PNs exhibit normal dendrite and axon targeting, suggesting that Dicer-2 is dispensable and the siRNA pathway is not required for PN targeting (Berdnik, 2008).
Next it was asked whether Dicer-2 could compensate for Dicer-1's function in PN targeting because their protein domain organization is highly similar. UAS-Dicer-2 was expressed in Dicer-1−/− PNs to test whether PN mistargeting phenotypes could be rescued as is the case for UAS-Dicer-1 expression. No alteration was seen in the Dicer1−/− dendrite mistargeting phenotypes in DL1 PNs, adPNs, or lPNs. This observation suggests that Dicer-2 cannot replace Dicer-1's function during PN targeting. It is proposed that Dicer-1-dependent PN targeting defects are caused by the absence of one or several miRNA(s), because Dicer-1, but not Dicer-2, is essential for miRNA-directed translation repression and mRNA turnover (Berdnik, 2008).
Many distinct mechanisms have been described for miRNA-mediated gene silencing. However, for all these, the RNA-induced silencing complex (RISC) containing the Argonaute (AGO) proteins as core components is required. AGO members can be divided into two groups, the ubiquitously expressed AGO and the reproductive cell-specific Piwi subfamily. The AGO subclass containing AGO1 and AGO2 in Drosophila is involved in small RNA loading into the RISC. Both miRNAs and siRNAs act as components of RISCs but use different silencing mechanisms. miRNAs typically contain several mismatches when paired with target mRNAs, causing mostly translational repression, whereas siRNAs are perfectly paired with target mRNAs leading to their degradation. AGO2 is described as a multiple-turnover RNA-directed RNA endonuclease acting in mRNA cleavage, whereas AGO1 functions in translational repression but also plays a role in efficient mRNA degradation. However, mRNAs targeted by almost perfectly paired miRNAs can also be degraded via AGO2. Thus, AGO1 is typically necessary for stable miRNA maturation and is essential for viability, whereas AGO2 is an essential component of the siRNA-directed RNA interference response (Berdnik, 2008).
To determine which AGO member is involved in PN targeting, MARCM clones of the strong loss-of-function allele AGO1k08121 and the AGO2414 null allele were examined. Surprisingly, normal PN dendrite and axon targeting were observed in AGO1k08121 and AGO2414 adPNs, and DL1 single neurons as dendrites elaborate in the single dorsolateral DL1 glomerulus like in WT. To test whether AGO1 and AGO2 could act in a redundant manner, PN clones were generated homozygous mutant for AGO1 in an AGO2 homozygous mutant background. 7/7 adPNs and 9/9 DL1 PNs exhibit normal targeting. In addition, axon-terminal arborization is normal in AGO1/AGO2 mutant DL1 cells (Berdnik, 2008).
There are several explanations for this surprising result. First, the AGO1k08121 allele may not be null. Second, perdurance of AGO1 protein from parental cells is capable of compensating for the loss of the AGO1 gene in homozygous mutant clones. AGO1k08121 mutants have drastically reduced mRNA levels, AGO1 is absent in homozygous AGO1k08121 embryo lysates, and AGO1k08121 has been shown to disrupt stable miRNA maturation. AGO1k08121 mutant wing disc clones miRNA function is disrupted as in pasha−/− and Dicer-1−/− clones as shown by using a bantam sensor transgene. Because of these facts and given that WT AGO1 mRNA or protein would be heavily diluted at least in neuroblast clones, the above two explanations imply that a very small amount of AGO1 would be sufficient for PN dendrite targeting. Third, perhaps one or more members of the Piwi subfamily thought to be expressed and function predominantly in the germline could compensate for the loss of AGO1/AGO2 in PNs. However, normal PN morphogenesis was observed in mutants for piwi1 and aubergineLL06590, and both are Piwi subfamily members. Lastly, PN dendrite targeting may utilize a novel miRNA-processing mechanism that is Dicer-1 dependent but AGO independent (Berdnik, 2008).
MicroRNA-mediated posttranslational regulation of gene expression has been documented in an increasing number of biological processes. Many miRNAs are developmentally regulated and show tissue-specific expression. In the nervous system, miRNAs have been shown to play roles during neurogenesis, specification of neuronal fate, neuronal morphogenesis, synaptogenesis, and neurodegeneration. This study has demonstrated a new function of the miRNA-processing pathway in regulating wiring specificity of the olfactory circuit (Berdnik, 2008).
The results support the model that one or more miRNA(s) are essential for regulating expression of genes that in turn regulate PN dendrite targeting and axon-terminal elaboration in identified neurons during development. Candidate target genes could be transcription factors that regulate wiring specificity in postmitotic neurons, cell-surface receptors for dendrite targeting, or their regulators. Expression or protein levels of such genes are essential for PN dendrite targeting. However, each miRNA is predicted to target hundreds of mRNAs and several miRNAs can regulate one mRNA, adding much more complexity to their regulatory function. Indeed, 7 miRNAs with available null mutants (out of 152 miRNAs predicted in the Drosophila genome were tested; none of them exhibit PN targeting defects. In flies, techniques that would allow the injection of individual or pools of mature miRNAs to rescue the neural phenotypes in pasha or Dicer-1 mutants, or mimic these phenotypes by injecting 'target protectors' that interfere with miRNA-mRNA interactions as in zebrafish, are currently not available. Therefore, it remains to be a future challenge to identify the miRNA(s), and ultimately their targets, for PN target selection. Looking for mutants with similar phenotypes as pasha and Dicer-1 in forward genetic screens or candidate gene approaches may help to identify specific miRNA and their targets (Berdnik, 2008).
Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. Stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. This study shows that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs (Friggi-Grelin, 2009).
cos2, fu, and smo mRNA can be regulated by a cluster of microRNAs, including miR-12 and miR-283, in Drosophila wing disc. The overexpression of this cluster decreases the levels of Smo, Cos2, and Fu proteins and activates the Hh pathway, as evidenced by the induction of dpp expression in the wing imaginal discs and by the adult wing outgrowth. The experiments presented in this study with the 3'-UTR sensors of smo, fu, or cos2 are in favor of a direct binding. To constitute a real proof of a direct effect, further experiments as direct biochemical binding assay or compensatory mutation between the 3'-UTR and the miRNAs will be necessary to perform (Friggi-Grelin, 2009).
Programs that have been created to genomewide predictions of Drosophila miRNA targets provide lists of presumptive miR-12, and miR-283 regulated genes. In addition to the current in vivo validations, miR-12 binding sites are predicted on the 3'-UTR of ci and no sites were found on the 3'-UTR of the Su(fu) gene. No decrease was observed in either of these two proteins in the microRNA cluster overexpressing clones. It is interesting to note that Su(fu) mRNA, encoding another negative regulator of Hedgehog signaling, has been shown to be targeted by miR-214 in zebrafish. Absence of miR-214 results in the reduction of muscle cell types, the specification of which is dependent on Hh pathway activity. Nevertheless, the current study shows that in Drosophila wing discs an absence of microRNA does not modify the Hh pathway, raising the question of what the role of microRNAs in Drosophila Hh pathway regulation is (Friggi-Grelin, 2009).
Could the microRNAs overexpression phenotype that was identified be artifactual and simply the result of forced overexpression of the microRNA cluster in a tissue in which it should be silent? It is thought that the answer is no, because Northern blot analysis and the increase of miR-sensor in the dcr-1 mutant clones showed that the microRNA cluster is indeed expressed in this tissue. This suggests that the cluster likely has a role in this tissue in which it is normally present. Is the microRNA cluster regulation of the cos2 and smo 3'-UTRs physiological? It is thought so, because an absence of either the microRNA cluster or of Dicer in the wing imaginal disc induces an increase in the Cos2- and Smo-sensor lines. This signifies that the microRNAs expressed from the cluster regulate the cos2 and smo 3'-UTRs and thus display some functionality in the disc during larval development. Altogether, these data clearly show that an artifactual situation in which the microRNA cluster is expressed in a tissue in which it should not be present has not been created. The miRs overexpression was also tested on embryonic patterning but it did not lead to any phenotype, suggesting that the miR cluster regulation on the Hh pathway is specific to larval tissues (Friggi-Grelin, 2009).
As miR-12 and miR-283, and likely redundant miRs, are present in every cell of the wing disc, one possibility is that their normal roles are to dampen down the levels of Hh pathway components, particularly Cos2 and Smo, to prevent the accidental activation or downregulation of the pathway. Indeed, expressing both the microRNA cluster and its targets in the same tissue could provide a means of 'buffering stochastic fluctuations' in mRNA levels or in protein translation rates within the Hh signaling pathway, as has been proposed for other processes (Friggi-Grelin, 2009).
The data possibly indicate that miRNAs are able to regulate two antagonistic components of the pathway, Cos2 and Smo. It has been shown that the stability of these two proteins is 'interdependent': an increased level of Cos2 in the wing imaginal disc lowers the level of Smo, and, in the opposite direction, increased Smo decreases the level of Cos2. It is proposed that the interregulation of Cos2/Smo levels is independent of their relative activities because Cos2 effect on Smo levels is observed in posterior cells in which Cos2 activity is strongly inhibited by the constitutive activation of the pathway. Therefore, eliminating the miRNA-mediated inhibition of Cos2 and Smo in Delta3miR or dcr-1 mutant cells likely initially increased the levels of both proteins, but then the resulting higher levels of each protein presumably downregulated the other; the net variation of Cos2 and Smo levels would therefore be null. This hypothesis is favored because the independent Smo- and Cos2-sensor lines, which are unaffected by this Cos2/Smo interregulation, showed increased levels of GFP staining in Delta3miR and dcr-1 mutant animals. This suggests that the levels of both Cos2 and Smo are increased in the mutant animals but, because of the downregulation of each protein by the other, no ultimate alterations in the levels of the proteins are observed. If so, an Hh phenotype would not be expected to be seen in the miR mutant (Friggi-Grelin, 2009).
The screen created a situation in which the expression of the microRNA cluster is deregulated, ultimately destabilizing Cos2 protein levels and thereby activating Ci and Hh target gene expression. Importantly, a similar situation might be encountered during tumoral development. Aberrant Hh signaling activity is known to trigger the development of diverse cancers. While several of these tumors have been linked to mutations in Hh signaling components, not all of them have, leaving open the possibility that they are caused by other factors such as microRNA misexpression. Interestingly, more than half of the known human microRNA genes are located near chromosomal breakpoints associated with cancer, and in some documented cases the microRNAs are amplified, leading to overexpression. Some upregulated microRNAs are possibly able to bind mRNAs encoding negative regulators of Hh signaling, such as Su(fu) or Ptc, and could thus induce the misactivation of the Hh pathway, as is observed in some cancers. Therefore, a fine analysis of microRNA expression levels and the levels of known Hh components should be considered in studies of Hh pathway-related cancers (Friggi-Grelin, 2009).
What does this study add to the current knowledge about miRNA regulation? The study shows that a cluster of three microRNAs can target several antagonistic components of the same pathway in vivo. This is novel and unexpected. This raises the question of how to interpret the miRNA expression signatures observed in human tumors. Indeed, as stated above, it has been proposed that miRNAs are differentially expressed in human cancers and contribute to cancer development. The working hypothesis in the cancer/miRNAs field is that key cancer genes are regulated by aberrant expression of miRNAs. The identification of a specific miRNA:mRNA interactor pair is generally accepted as being of biological importance when the mRNA encodes a tumor suppressor or an oncogene whose expression is modified in the tumor. This study shows indirectly that this is an oversimplified view, because identifying an oncogene or tumor suppressor as a target of a miRNA may not provide a full explanation for tumor development if the same miRNA hits other antagonistic components of the same pathway that nullify the effect of the identified miRNA:mRNA interactor pair (Friggi-Grelin, 2009).
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