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).
Basyuk, E., Suavet, F., Doglio, A., Bordonne, R. and Bertrand, E. (2003). Human let-7 stem-loop precursors harbor features of RNase III cleavage products. Nucleic Acids Res. 31(22): 6593-7. 14602919
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818): 363-6. 11201747
Boutla, A.,, (2001). Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. Curr. Biol. 11: 1776-1780. 11719220
Cerutti, L., Mian, N., and Bateman, A. (2000). Domains in gene silencing and cell differentiation proteins: The novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25: 481-482. 11050429
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. and Hannon, G. J. (2004). Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014): 231-5. 15531879
Doi, N., et al. (2003). Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr. Biol. 13: 41-46. 12526743
Elbashir, S. M., Lendeckel, W. and Tuschl, T. (2001a). RNA interference is mediated by 21 and 22 nt RNAs. Genes Dev. 15: 188-200. 11157775
Elbashir, S. M., et al. (2001b). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20: 6877-6888. 11726523
Filippov, V., Solovyev, V., Filippova, M. and Gill, S. S. (2000). A novel type of RNase III family proteins in eukaryotes. Gene 245(1): 213-21. 10713462
Findley, S. D., Tamanaha, M., Clegg, N. J. and Ruohola-Baker, H. (2003). Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage. Development 130: 859-871 . 12538514
Forstemann, K., Tomari, Y., Du, T., Vagin, VV., Denli, A. M., Bratu, D. P., Klattenhoff, C., Theurkauf, W. E. and Zamore, P. D. (2005). Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3(7): e236. 15918770
Giraldez, A. J., et al. (2005). MicroRNAs regulate brain morphogenesis in zebrafish. Science 308(5723): 833-8. 15774722
Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T. and Doratotaj, B. (2004). The Microprocessor complex mediates the genesis of microRNAs. Nature 432: 235-240. 15531877
Gregory, R. I., et al. (2005). Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123: 631-640. 16271387
Grishok, A., et al. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106(1): 23-34. 11461699
Hammond, S. M., Bernstein, E., Beach, D. and Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404(6775): 293-6. 10749213
Hatfield, S. D. et al. (2005). Stem cell division is regulated by the microRNA pathway. Nature 435(7044): 974-8. 15944714
Hutvagner, G., et al. (2001). A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293: 834-838. 11452083
Hutvagner, G. and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297: 2056-2060. 12154197
Ishizuka, A., Siomi, M. C. and Siomi1, H. (2002). A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16: 2497-2508. 12368261
Jacobsen, S. E., Running, M. P. and Meyerowitz, E. M. (1999). Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. Development 126(23): 5231-43. 10556049
Jiang, F., et al. (2005). Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev. 19: 1674-1679. 15985611
Jin, Z. and Xie, T. (2007). Dcr-1 maintains Drosophila ovarian stem cells. Curr. Biol. 17(6): 539-44. Medline abstract: 17306537
Jing, Q., et al. (2005). Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120(5): 623-34. 15766526
Kanellopoulou, C., et al. (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19(4): 489-501. 15713842
Kataoka Y., Takeichi M. and Uemura T. (2001) Developmental roles and molecular characterization of a Drosophila homologue of Arabidopsis Argonaute1, the founder of a novel superfamily. Genes Cells 6: 313-325. 11318874
Ketting, R. F., et al. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans Genes Dev. 15: 2654-2659. 11641272
Knight, S. W. and Bass, B. L. (2001). A role for the RNase III enzyme DCR-1 in RNA Interference and germ line development in Caenorhabditis elegans. Science 293: 2269-2271. 11486053
Landthaler, M., Yalcin, A. and Tuschl, T. (2004). The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14(23): 2162-7. 1558916
Lee, Y., Ahn, C., Han, J., Choi, H. and Kim, J. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425: 415419. 14508493
Lee, Y. S., Nakahara, K., Pham, J. W. Kim, K., He, Z., Sontheimer, E. J. and Carthew, R. W. (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117: 69-81. 15066283
Lipardi, C., Wei, Q. and Paterson, B. M. (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107: 297-307. 11701121
Megosh, H. B., Cox, D. N., Campbell, C. and Lin, H. (2006). The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr. Biol. 16(19): 1884-94. Medline abstract: 16949822
Motamedi, M. R., et al. (2004). Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119(6):789-802. 15607976
Murchison, E. P., et al. (2007). Critical roles for Dicer in the female germline. Genes Dev. 21(6): 682-93. Medline abstract: 17369401
Nakahara, K., Kim, K., Sciulli, C., Dowd, S. R., Minden, J. S. and Carthew, R. W. (2005). Targets of microRNA regulation in the Drosophila oocyte proteome. Proc. Natl. Acad. Sci. 102(34): 12023-8. 16099838
Nicholson, R. H. and Nicholson, A. W. (2002). Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference. Mamm Genome 13(2): 67-73. 11889553
Noma K., et al. (2004). RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat Genet. 36(11): 1174-80. 15475954
Okamura, K., Hagen, J. W., Duan, H., Tyler, D. M. and Lai, E. C. (2007). The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130(1): 89-100. Medline abstract: 17599402
Park, W., et al. (2002). CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12: 1484-1495. 12225663
Pasquinelli, A. E., et al. (2000). Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408(6808): 86-9. 11081512
Preall, J. B., He, Z., Gorra, J. M. and Sontheimer, E. J. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol. 16(5): 530-5. 16527750
Provost, P., Dishart, D., Doucet, J., Frendewey, D., Samuelsson, B., and Radmark, O. (2002). Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 21: 5864-5874. 12411504
Ray, A., Lang, J. D., Golden, T. and Ray, S. (1996). SHORT INTEGUMENT (SIN1), a gene required for ovule development in Arabidopsis, also controls flowering time. Development 122(9): 2631-8. 8787738
Ray, S., Golden, T. and Ray, A. (1996). Maternal effects of the short integument mutation on embryo development in Arabidopsis. Dev. Biol. 180(1): 365-9. 8948599
Rose, D.H. Kim, M. Amarzguioui, J.D. Heidel, M.A. Collingwood, M.E. Davis, J.J. Rossi and M.A. Behlke (2005). Functional polarity is introduced by Dicer processing of short substrate RNAs, Nucleic Acids Res. 33: 4140-4156. 16049023
Saito, K., Ishizuka, A., Siomi, H., Siomi, M. C. (2005). processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol. 3(7): e235. 15918769
Schmidt, A., Palumbo, G., Bozzetti, M. P., Tritto, P., Pimpinelli, S. and Schafer, U. (1999). Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster. Genetics 151: 749-760. 9927466
Schramke, V. and Allshire, R. (2003). Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science 301: 1069-1074. 12869699
Sigova, A., Rhind, N. and Zamore, P. D. (2004). A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev. 18(19): 2359-67. 15371329
Sugiyama, T., Cam, H., Verdel, A., Moazed, D. and Grewal, S. I. (2005). RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. Proc. Natl. Acad. Sci. 102(1):152-7. 1561584
Tabara, H., Yigit, E., Siomi, H. and Mello. C. C. (2002). The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109: 861-871. 12110183
Verdel A., et al. (2004). RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303(5658): 672-6. 14704433
Wilson, J. E., Connell, J. E. and Macdonald, P. M.(1996). aubergine enhances oskar translation in the Drosophila ovary. Development 122: 1631-1639. 8625849
Xie, Z., Kasschau, K. D. and Carrington, J. C. (2003). Negative feedback regulation of Dicer-Like1 in Arabidopsis by microRNA-guided mRNA degradation. Curr. Biol. 13: 784-789. 12725739
Zeng, Y., Yi, R. and Cullen, B. R. (2005), Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 24(1): 138-48. 15565168
Zhang, H., et al. (2004). Single processing center models for human Dicer and bacterial RNase III. Cell 118(1): 57-68. 15242644
date revised: 20 December 2007
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