The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. This study characterizes Drosophila small RNAs that derive from short intronic hairpins termed 'mirtrons.' Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and evidence is provided that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs (Okamura, 2007).
This study has characterized a class of intronic hairpins, termed mirtrons, that generate ~22 nt regulatory RNAs in Drosophila. The biogenesis of mirtrons is distinct from that of canonical miRNAs. Although alternate mechanisms are not excluded, the data points to a mechanism in which mirtron maturation bypasses cleavage by the pre-miRNA-generating enzyme Drosha but is instead initiated by splicing and intron lariat debranching. This differs explicitly from the processing of canonical intronic miRNA genes, whose cleavage by Drosha occurs prior to host intron splicing. However, the mirtron pathway merges with the canonical miRNA pathway to generate active regulatory RNAs, since debranched mirtrons are productive substrates of Exportin-5 and the Dicer-1/loqs system, yielding small RNAs that can repress target transcripts. This study showed specifically that mirtron-derived small RNAs can associate with Ago1 and require Ago1 to regulate seed-matched targets (Okamura, 2007).
The functional similarity between mirtrons and miRNA precursors is bolstered by the observation that miR-10-3p and the small-RNA product of a mirtron hairpin in Vha-SFD are extensively related across their 5' halves, are derived from the same (right-hand) hairpin arm, are the most abundant products of their respective hairpins, and have the same seed (positions 2-8, AAAUUCG). The small-RNA products of mirtrons are catagorized as a novel subclass of miRNAs (Okamura, 2007).
Fourteen mirtron loci were identified from a high-throughput sequencing effort that confidently identified 133 canonical miRNA genes; thus, mirtrons constitute a considerable fraction of total miRNA genes in Drosophila. In contrast, while a majority of canonical miRNA genes are well-conserved among the sequenced Drosophilids, most mirtrons arose recently during evolution. Since newly evolved miRNAs are thought to have fewer targets than highly conserved miRNAs, the regulatory networks involving mirtrons may be proportionally smaller than those mediated by canonical miRNAs. Still, the findings that both 'old' and 'young' mirtrons (1) produce miRNAs that associate with Ago1, (2) can actively repress minimally paired seed targets, and (3) display patterns of divergence on microevolutionary scales that indicate their incorporation into endogenous regulatory networks together suggest that mirtrons exert appreciable effects on biological networks. Indeed, the relative ease with which mirtrons have been born and/or lost raises the intriguing possibility that the changing mirtronic content of Drosophila genomes has contributed to fly speciation (Okamura, 2007).
The existence of mirtrons has implications for the interpretation of miRNA genetics. It is now recognized that the Dicer mutant condition does not solely reflect the loss of miRNAs, since Dicer has additional roles in chromatin dynamics and/or processing of exogenous or other endogenous dsRNA, depending on the organism. Drosha mutant cells do not accurately reflect the loss of miRNAs either; since Drosha processes other ncRNAs, including rRNAs. It has been suggested that DGCR8/Pasha mutant cells more purely reflect a 'miRNA null' state. This may not be the case either, because the mirtron pathway generates a subclass of miRNAs via a nuclear pathway that is largely, if not completely, distinct from the microprocessor. Therefore, caution should be exercised when using processing-enzyme mutants to assess the contribution of small RNAs to a given biological process (Okamura, 2007).
The data demonstrate that the Drosophila mirtron pathway merges the splicing/debranching pathway with the dicing pathway to generate functional miRNAs. Since the key parts of this hybrid small-RNA pathway are deeply conserved mechanisms for RNA processing, it seems plausible that mirtrons may exist outside of Drosophila. Since debranched introns are normally quite labile; however, it is hypothesize that critical to the operation of the mirtron pathway is a dedicated mechanism to hand-off debranched introns to the hairpin export machinery. Having such a mechanism in place may prove key to the existence of mirtrons in other species (Okamura, 2007).
It was asked if Loqs forms a complex in vivo with Dicer-1. For these studies, Dicer-1 tagged with the Flag epitope and Loqs tagged with the myc epitope were simultaneously expressed in S2 cells. Then Dicer-1 was immunoprecipitated with anti-Flag antibodies, and Loqs with anti-myc antibody and then the precipitates were analyzed by immunoblotting. In reciprocal assays, Dicer-1 and Loqs were found to co-precipitate. Consistent with these findings that Dicer-1 and Loqs form a complex in vivo, both proteins are localized predominantly in the cytoplasm of S2 cells (Saito, 2005).
It was further investigated whether Loqs can bind to Dicer-1 in vitro. Dicer-1 was produced by an in vitro translation system and used in binding assays with recombinant Loqs fused to glutathione S-transferase (GST). GST-Loqs interacts with Dicer-1 even in the presence of RNase A, whereas GST itself shows no detectable binding. These results demonstrate that the association of Loqs with Dicer-1 occurs both in vivo and in vitro, and that RNA molecules do not appear to mediate the association (Saito, 2005).
To examine the functional connection between the Dicer-1-Loqs complex and pre-miRNA processing, whether depletion of Dicer-1 or Loqs has any effect on the production of mature miRNA from the precursor was investigated. First whether cytoplasmic lysates of S2 cells are capable of processing synthetic Drosophila let-7 precursor RNA into functional mature let-7 was investigated. In this experiment, the synthetic let-7 precursor RNA was converted to mature let-7 in S2 cytoplasmic lysates, as is the case in embryo lysates. In an in vitro RNAi assay, target RNA harboring a sequence perfectly complementary to mature let-7 was cleaved efficiently within the let-7 complementary sequence, thus showing production of functional let-7 in S2 cell lysates. Cytoplasmic lysates from Dicer-1- or Loqs-depleted cells were then subjected to the pre-let-7 processing assay. Both Dicer-1 and Loqs depletion led to reductions of mature let-7 compared with controls, showing that both Dicer-1 and Loqs function in pre-miRNA processing (Saito, 2005).
Next, pre-miR-bantam was used as a substrate for pre-miRNA processing assays. It has been shown that S2 cell extracts contain primary-miRNA processing activity that cleaves pri-miRNA into an approximately 60- to 70-bp pre-miRNA precursor. This processing is known to occur in the nucleus; thus pre-miR-ban was prepared by in vitro processing of pri-miR-ban incubated with S2 nuclear extracts. Uniformly labeled pre-miR-ban was then gel-purified and used as a substrate for analysis of pre-miRNA processing. Incubation of the pre-miRNA with S2 cytoplasmic extracts results in the appearance of a mature 21-nucleotide miR-ban. Then the requirement of Dicer-1 and Loqs in pre-miR-ban processing was examined. Incubation of pre-miRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic extracts results in a marked reduction in mature miRNA levels. In contrast, depletion of Dicer-2 or R2D2 shows no measurable reduction of mature miRNA levels. Then the pre-miRNA processing activity of the purified complexes (both Flag-Dicer-1 and Flag-Loqs complexes) was assayed. That the Flag-Loqs complex contains Dicer-1 was confirmed by immunoblotting. Both Dicer-1 and Loqs complexes are capable of generating maturemiR-ban from pre-miR-ban. Several steps in the RNAi and miRNA pathways are known to require a divalent metal ion. In addition, it is well known that RNase III-type enzymes require divalent metals for cleavage. Flag-Dicer-1 complex was employed and the processing was performed in the presence of magnesium ions or EDTA in a buffer. No pre-miRNA processing activity is detected at 10 mM EDTA. These results demonstrate that the Dicer-1-Loqs complex converts pre-miRNAs into mature miRNAs in a divalent metal ion-dependent manner (Saito, 2005).
To further examine the requirement for Loqs in pre-miRNA processing, Flag-Dicer-1 complex was purified under a harsher condition (high salt), where Dicer-1 is stripped of most Loqs protein, and this Dicer-1 complex was used in pre-miRNA processing assays with or without supplement of recombinant GST-Loqs. Without any supplement, the Flag-Dicer-1 complex purified under the harsh condition showed less activity than that under mild condition. Then GST-Loqs was added in the assay mixture. The addition of GST-Loqs to the Dicer-1 complex stimulates the processing of pre-miRNA. GST-Loqs alone does not show any significant pre-miRNA processing activity. These results show that Loqs is required for stimulating the processing of pre-miRNAs. Interestingly, it was found that the Dicer-1 complex purified under the harsh condition displays considerable siRNA-generating activity on the long dsRNA substrate in vitro, although previous genetic studies have shown that Dicer-1 is not required for siRNA production. The addition of GST-Loqs inhibits this effect. Western blot analysis shows that the Dicer-1 complex used in this experiment does not contain appreciable Dicer-2. GST-Loqs alone shows no activity for generating siRNAs from long dsRNAs. These results suggested that Dicer-1, stripped of much of its bound Loqs, processes both dsRNA and pre-miRNA substrates, but re-addition of recombinant Loqs suppresses dsRNA processing activity and enhances pre-miRNA processing activity. These findings thus imply that much of the apparent substrate specificity of Dicer-1 in vivo results from its association with Loqs. Although very unlikely, it is, however, formally possible that the Dicer-1 immunoprecipitates may contain very small amounts of Dicer-2 protein that can catalyze long dsRNA cleavage, and that addition of a large amount of dsRBD-containing Loqs may block the activity of Dicer-2 in this experiment (Saito, 2005).
The presence of endogenous miRNA was examined in RNA preparations from Flag-Dicer-1 and Flag-Loqs complexes obtained from S2 cells using anti-Flag antibodies. The Dicer-1 complex contains both the pre- and mature form of miR-ban, and the complex seems to preferentially bind the precursor form of miR-ban. In contrast, the precursor form of miR-ban is barely detectable in the Loqs complex, though it contains mature miR-ban. However, EDTA treatment, which inhibits pre-miRNA processing activity, results in an accumulation of pre-miR-ban in the Loqs complex. This may suggest that part of Flag-tagged Loqs protein interacts with Dicer-1 or pre-miRNAs or both. Alternatively, Flag-Loqs complexes may rapidly process pre-miRNAs into mature miRNAs and, therefore, may only transiently interact with them. Nonetheless, these results suggest that Dicer-1-Loqs complexes associate with both pre- and mature miRNAs in vivo (Saito, 2005).
Argonaute protein AGO1 is required for stable production of mature miRNAs and associates with Dicer-1. Thus, attempts were made to ascertain if Loqs is also present in an AGO1-associated complex, and if so, if the AGO1 complex is capable of processing pre-miRNA in vitro. Flag-Loqs and AGO1 tagged with TAP were simultaneously expressed in S2 cells, and the AGO1-TAP complex was purified through immunoglobulin G (IgG) bead-binding. The IgG bound was then subjected to Western blot analysis using anti-Dicer-1, anti-AGO1, or anti-Flag (for Loqs detection) antibodies. Not only Dicer-1 but also Loqs was detected in the AGO1 complex. These results indicate that all three proteins are present in the same complex, although they cannot exclude the possibility that there is one complex that contains AGO1 and Dicer-1 but not Loqs, and another complex that contains AGO1 and Loqs but not Dicer-1. The pre-miRNA processing activity of the AGO1 complex was then examined. Pre-miR-ban was utilized as a substrate. The AGO1 complex is able to efficiently process pre-miR-ban into the mature form. In contrast, another Argonaute protein AGO2-associated complex shows no such activity, which is consistent with the finding that the AGO2-associated complex does not contain Dicer-1. Considered together, these results showed that Dicer-1 and Loqs form a functional complex that mediates the genesis of mature miRNAs from pre-miRNAs, and suggested that the resultant mature miRNAs are loaded onto an AGO1-associated complex, which probably is miRNA-associated RISC, through specific interaction of AGO1 with Dicer-1 and Loqs (Saito, 2005).
Reduction of R2D2 protein by RNAi destabilizes Dcr-2; conversely, RNAi of Dcr-2 renders R2D2 unstable. In contrast, RNAi of loqs in S2 cells reduced Dcr-1 protein levels by no more than 15%, suggesting that Loqs functions together with Dcr-1 in pre-miRNA processing, rather than that Loqs is simply needed to stabilize Dcr-1 protein. However, loqsf00791 mutant ovaries, which lack detectable Loqs protein, contain 70% less Dcr-1 than wild-type. A role for Loqs in both Dcr-1 function and in Dcr-1 stability suggests that the two proteins physically interact, like R2D2 and Dcr-2. Therefore, tests were performed to see if Dcr-1 and Loqs are components of a common complex (Forstemann, 2005).
Myc-tagged versions for two protein isoforms of Loqs, Loqs PA and Loqs PB, were expressed in S2 cells, and the tagged proteins were immunoprecipitated with anti-myc monoclonal antibodies. The immunoprecipitated protein was analyzed by Western blotting using a polyclonal anti-Dcr-1 antibody. Dcr-1 protein co-immunoprecipitates with myc-tagged Loqs. When myc-tagged GFP was expressed in place of myc-tagged Loqs, no Dcr-1 protein was recovered in the anti-myc immunoprecipitate. Similarly, an affinity purified, polyclonal antibody directed against the N-terminus of endogenous Loqs protein also co-immunoprecipitated Dcr-1 protein. This interaction is resistant to treatment with RNase A. No co-immunoprecipitation of Dcr-2 with myc-tagged Loqs PB was detected under conditions where Dcr-1 was readily detected, but it cannot be excluded that Dcr-2 is a substoichiometric component of a complex that contains both Dcr-1 and Loqs (Forstemann, 2005).
When immunoprecipitated with anti-Dcr-1 antibody, both myc-tagged Loqs protein isoforms-PA and PB-associate with Dcr-1. Moreover, the antibody against endogenous Loqs protein detected two bands corresponding in size to Loqs PA and Loqs PB in the proteins immunoprecipitated with the anti-Dcr-1 antibody. Loqs PB comprises only approximately 22% of the total Loqs protein in S2 cells, but corresponds to approximately 95% of the Loqs associated with Dcr-1. Loqs PA, which is expressed at comparable levels in S2 cells, accounts for most of the remaining Loqs associated with Dcr-1. In contrast, the putative Loqs PC protein comprises the majority of S2 cell Loqs, but is not recovered in the Dcr-1 immunoprecipitate. Intriguingly, Loqs PA and PB contain a third dsRBD that Loqs PC lacks; perhaps this third dsRBD is required for the association of Loqs with Dcr-1 (Forstemann, 2005).
The immunoprecipitated Dcr-1-Loqs complexes accurately convert pre-miRNA to mature miRNA. Pre-miRNA processing by the immunoprecipitates is efficient and accurate when the anti-Dcr-1 antibody was used, and also when anti-myc antibody and myc-tagged Loqs was used. Thus, Dcr-1 and Loqs co-associate in a complex capable of converting pre-miRNA into mature miRNA. The data also demonstrate that an N-terminal tandem myc tag does not perturb Loqs function in pre-miRNA cleavage (Forstemann, 2005).
Next, the size of the pre-miRNA processing complex was estimated by gel filtration chromatography. Pre-miRNA processing activity chromatographs as a broad approximately 525-kDa peak that overlaps the peaks of both Dcr-1 and Loqs proteins. Dcr-1 protein chromatographs as an approximately 480-kDa complex that overlaps the peak of Loqs PB, which chromatographs as an approximately 630-kDa complex. The Loqs PB isoform accounts for most of the Dcr-1-associated Loqs in S2 cells. The apparent size of the Dcr-1 complex suggests that it is either associated with proteins in addition to Loqs or that the complex has an elongated shape that increases its apparent molecular weight. Pre-miRNA processing activity, Loqs, and Dcr-1 were all well resolved from the approximately 230-kDa peak of Dcr-2, which corresponds to the Dcr-2/R2D2 heterodimer. Although the peaks of Loqs and Dcr-1 do not co-migrate, Dcr-1 is stably associated with Loqs after gel filtration: Dcr-1 and Loqs reciprocally co-immunoprecipitates from the pooled peak Dcr-1 fractions. Loqs was not detected in the Dcr-2 peak by this method. Loqs PC, which does not associate with Dcr-1 in immunoprecipitation, chromatographs as a 58-kDa protein, suggesting that it is a free monomeric protein (Forstemann, 2005).
Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, that arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. This study shows that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2. Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles (Czech, 2008).
Drosophila expresses five Argonaute proteins, which segregate into two classes. The Piwi proteins (Piwi, Aubergine and AGO3) are expressed in gonadal tissues and act with Piwi-interacting RNAs (piRNAs) to suppress mobile genetic elements. The Argonaute class contains AGO1 and AGO2. AGO1 binds microRNAs (miRNAs) and regulates gene expression. The endogenous binding partners of AGO2 have remained enigmatic (Czech, 2008).
Transgenic flies were generated expressing epitope-tagged AGO2 under the control of its endogenous promoter. Tagged AGO2 localizes to the cytoplasm of germline and somatic cells of the ovary. Immunoprecipitated AGO2-associated RNAs differ in their mobility from those bound to AGO1. Deep sequencing of small RNAs from AGO1 and AGO2 complexes yielded 2,094,408 AGO1-associated RNAs and 916,834 AGO2-associated RNAs from Schneider (S2) cells, and 455,227 AGO2-associated RNAs from ovaries that matched perfectly to the Drosophila genome. Three libraries were sequenced derived from 18-29-nucleotide RNAs (936,833 sequences from wild-type ovaries, 1,042,617 sequences from Dicer-2 (Dcr-2) mutant ovaries, and 1,946,339 sequences from loquacious (loqs) mutant ovaries) and an 18-24-nucleotide library from wild-type testes (522,848 sequences). Finally, the analysis included 92,363 published sequences derived from 19-26-nucleotide RNAs from S2 cells (Czech, 2008).
Among the ~50% of AGO2-associated RNAs from S2 cells that did not match the genome, ~17% matched the flock house virus (FHV), a pathogenic RNA virus and reported target for RNAi in flies. These probably arose because of persistent infection of the S2 cultures (Czech, 2008).
After excluding presumed degradation products of abundant cellular RNAs, each of the total RNA libraries were divided into two categories: annotated miRNAs and the remainder. For the S2 cell library, the size distribution of these populations formed two peaks, with non-miRNAs lying at 21 nucleotides and miRNAs exhibiting a broader peak from 21 to 23 nucleotides. Libraries derived from AGO1 and AGO2 complexes almost precisely mirrored these two size classes. In the ovary library, this approach revealed three size classes. Whereas two reflected those seen in S2 cells, a third class comprised piRNAs. Again, RNA size profiles from AGO2 or Piwi family immunoprecipitates mirrored those within the total ovary library. These data demonstrate that AGO2 is complexed with a previously uncharacterized population of small RNAs (Czech, 2008).
Whereas known miRNAs comprised more than 97% of AGO1-associated RNAs in S2 cells, they made up only 8% or 20% of the AGO2-bound species in S2 cells or ovaries, respectively. The remaining small RNAs in AGO2 complexes formed a complex mixture of endogenous siRNAs (endo-siRNAs). Among these, transposons and satellite repeats contributed substantially to AGO2-associated small RNAs in S2 cells (27%) and ovaries (53%). The nature of the transposons giving rise to abundant siRNAs in ovaries and S2 cells differed substantially, probably reflecting differential expression of specific transposons in these tissues. Unlike piRNAs, neither somatic nor germline siRNAs exhibited a pronounced enrichment for sense or antisense species (Czech, 2008).
In accord with these findings, knockdown of AGO2 in S2 cells leads to increased expression of several mobile elements. In the germ line, the Piwi-piRNA system has been reported as the dominant transposon-silencing pathway. Nevertheless, several transposons, with a potential to be targeted by siRNAs, were substantially derepressed in AGO2 mutant or Dcr-2 mutant ovaries. Although comparisons of relative abundance were difficult, both piRNAs and siRNAs mapped to piRNA clusters, with the regions that generate uniquely mapping species generally overlapping. Thus, piRNA loci are a possible source for antisense RNAs matching transposons and might serve a dual function in small RNA generation. Considered together, these data suggest that endo-siRNAs repress the expression of mobile elements, in some tissues acting alongside piRNA pathways (Czech, 2008).
To probe the nature of the remaining endo-siRNAs, genomic sites, which give rise to multiple uniquely mapping RNAs that do not fall into heterochromatic regions, were computationally extracted. These generally segregated into two categories, termed structured loci or convergently transcribed loci (Czech, 2008).
Transcripts from structured loci can fold to form extensive double-stranded RNA directly. The two major loci, termed esi-1 and esi-2, gave rise to half of the 20 most abundant endo-siRNAs in ovaries and also generated siRNAs in embryos, larvae and adults. esi-1, annotated as CG18854, can produce an ~400-base pair (bp) dsRNA through interaction of its 5' and 3' untranslated regions. esi-2 overlaps with CG4068 and consists of 20 palindromic ~260-nucleotide repeats. All siRNAs derived from these two loci arise from one genomic strand. In some previously characterized instances (for example, Arabidopsis trans-acting-siRNAs) Dicer generates 'phased' siRNAs with 5' ends showing a 21-nucleotide periodicity. In all tissues examined, esi-1 and esi-2 produced phased siRNAs, consistent with a defined initiation site for Dicer processing. Phasing was not observed for viral or repeat-derived siRNAs. Finally, siRNAs from both loci also joined AGO1 in proportions greater than siRNAs produced from transposons and repeats, perhaps owing to the imperfect nature of the dsRNA that they produce (Czech, 2008).
AGO2 regulates gene expression by cleavage of complementary sites rather than by recognition of seed sites typical of AGO1-miRNA-mediated regulation. Possible targets of endo-siRNAs were sought by identifying transcripts with substantial complementarity. A highly abundant siRNA from esi-2 is highly complementary to the coding sequence of the DNA-damage-response gene mutagen-sensitive 308 (mus308). Using a modified rapid amplification of cDNA ends (RACE) protocol, mus308 fragments were detected with 5' ends corresponding precisely to predicted endo-siRNA cleavage sites. Moreover, AGO2 and Dcr-2 loss consistently increased mus308 expression in testis and to a lesser extent in ovaries, consistent with the relative abundance of esi-2 siRNAs in these tissues. Finally, a reporter gene containing two mus308 target sites was significantly derepressed in S2 cells on depletion of Dcr-2 or AGO2 but not of Dcr-1 or AGO1. Although extensive complementarity between other endo-siRNAs and messenger RNAs was rare, several esi-1-derived siRNAs complementary to CG8289 were found, suggesting a potential regulatory interaction in vivo (Czech, 2008).
A second group of siRNA-generating loci contained regions in which dsRNAs can arise from convergent transcription. If sorted for siRNA density, most of the top 50 ovarian and S2 cell siRNA loci lay in regions where annotated 3' UTRs or expressed-sequence-tags corresponding to convergently transcribed protein-coding genes overlap. Typically, siRNAs arise on both genomic strands but only from overlapping portions of convergent transcripts. Examining all 998 convergently transcribed gene pairs in the Drosophila genome with annotated overlapping transcripts, the peak abundance of ovarian siRNAs was found to be at the centre of the overlap, with sharp declines away from this region. In an alternative arrangement, Pgant35A produces sense and antisense siRNAs across its entire annotated transcript, consistent with expressed-sequence-tag support for antisense transcription traversing this locus (Czech, 2008).
Thus, a large number of Drosophila genes generate endogenous siRNAs, with most having perfect complementarity to the 3' UTRs of neighbouring genes. Relative levels of endo-siRNAs generated from each convergent transcription unit were low, and no or little change (up to a ~1.3-fold increase) was found in the expression of such genes in AGO2 mutant ovaries. Possibly, the level of small RNAs produced by this genomic arrangement is inconsequential, amounting to noise within silencing pathways. However, there are probably circumstances wherein regulation by such arrangements might substantially impact expression (Czech, 2008).
In S2 cells, two neighbouring loci encoded nearly 16% of AGO2-associated RNAs. These reside within a large intron of klarsicht and did not generate siRNAs in any other tissue. A similar locus, corresponding to CG14033, was found within an intron of thickveins and gave rise to testis-specific siRNAs. Although the function of both siRNA clusters is unclear, the thickveins cluster shares considerable complementarity to CG9203, and loss of AGO2 and Dcr-2 mildly increased CG9203 mRNA levels in testis but not in ovaries (Czech, 2008).
Dcr-2 has been implicated in the production of siRNAs from viral replication intermediates or exogenously introduced dsRNAs, whereas Dcr-1 has been linked to miRNA biogenesis. In agreement with these observations, all endo-siRNA classes were lost in Dcr-2 mutant ovaries. To obtain more insight into the genetic requirements for endo-siRNA biogenesis and stability, components of siRNA and miRNA pathways were depleted in S2 cells, and levels of abundant siRNAs derived from structured loci were analysed . Although depletion of Dcr-2 and AGO2 resulted in substantial reductions in siRNA levels, little or no changes were observed on Drosha, Pasha, Dcr-1 or AGO1 depletion. Unexpectedly, virtually no requirement was found for the Dcr-2 partner R2D2 but a strong requirement was found for the Dcr-1 partner Loquacious. Only one analysed siRNA exhibited partial dependence on R2D2, potentially correlating with the extensive dsRNA character of its precursor duplex. Artificial sensors for endo-siRNAs from esi-1 and esi-2 in S2 cells gave patterns of de-repression that matched analysis of endo-siRNA levels (Czech, 2008).
Analysis of the most abundant siRNA from esi-2 in flies mutant for Dcr-2, AGO2, r2d2 or loqs extended these findings from cell culture. To examine the unexpected requirement for loqs more broadly, small RNAs were sequenced from loqs-mutant ovaries and a near complete loss of endo-siRNAs from structured loci was observed. A much smaller impact of loqs was seen on endo-siRNAs derived from repeats and convergent transcription units. However, an involvement of Loqs and not R2D2 in the function of siRNAs derived from perfect dsRNA precursors was supported by analysing the impact of depleting siRNA/miRNA pathway components on the ability to suppress FHV replication in infected S2 cell cultures (Czech, 2008).
The results uncover an unanticipated role for Loqs in siRNA biogenesis and suggest that R2D2 has a lesser impact on at least two types of endogenous siRNAs. It is well established that Loqs partners with Dcr-1 for miRNA processing. To probe a molecular interaction with Dcr-2, Loqs binding partners were analyzed using quantitative proteomics. Dcr-1 and Dcr-2 were both abundant in Loqs immunoprecipitates from cultured cells and flies, supporting a physical interaction between Dcr-2 and Loqs (Czech, 2008).
Among animals, endo-siRNA pathways have so far been restricted to Caenorhabditis elegans. The current results extend the prevalence of such systems to Drosophila and parallel recent discoveries of an endo-siRNA pathway in mouse oocytes. These systems have many common features but also key differences. In both, siRNAs collaborate with piRNAs to repress transposons. Also, mouse and Drosophila both generate endo-siRNAs from structured loci. In mouse, dsRNAs can form by pairing of sense protein-coding transcripts with antisense transcripts from pseudogenes. Whether or not transcripts from unlinked sites lead to siRNA production in Drosophila is unclear. However, transposon sense transcripts may hybridize to antisense sequences transcribed from piRNA clusters to form endo-siRNA precursors. In flies, a much larger number of genic loci enter the pathway as compared to mice because convergent transcription of neighbouring genes frequently creates overlapping transcripts. Overall, annotation of the Drosophila genome indicates that a significant proportion is transcribed in both orientations, providing widespread potential for dsRNA formation. This property is shared by many other annotated genomes, raising the possibility that the RNAi pathway has broad impacts on gene regulation. Viewed in combination, these studies suggest an evolutionarily widespread adoption of dsRNAs as regulatory molecules, a property previously ascribed only to miRNAs (Czech, 2008).
Drosophila expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ~22-23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ~24-28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ~21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNAbinding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. EndosiRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively (Zhou, 2009).
A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).
This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).
The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).
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