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).
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