Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila (original) (raw)
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Cell, 2004
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MicroRNAs in Drosophila Development
International Review of Cell and Molecular Biology, 2011
7.7. miRNAs involved in wingless/Wnt signaling 45 7.8. A miRNA in Notch signaling 46 7.9. A miRNA that regulates nuclear receptor signaling 46 7.10. miRNAs that regulate Hox-cluster genes 47 7.11. A miRNA that regulates circadian rhythm 8. Concluding Remarks 48 Acknowledgment 49 References 50
Cell, 2007
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1-or Ago2containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNAbinding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.
The Mirtron Pathway Generates microRNA-Class Regulatory RNAs in Drosophila
Cell, 2007
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. In this study, we characterize 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 we provide evidence 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.
Targets of microRNA regulation in the Drosophila oocyte proteome
Proceedings of the National Academy of Sciences, 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, we compared the proteome from wild-type oocytes 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.
Cell, 2005
biological function of miRNAs is still very limited, and 2 Laboratory of RNA Molecular Biology few mRNA targets have been validated in vivo. Rockefeller University In plants, most mRNA targets are readily identifiable 1230 York Ave due to near-complete sequence complementarity, and New York, New York 10021 miRNAs appear to act mostly by driving degradation of 3 Department of Systems Biology the target mRNA. This has greatly facilitated the func-Harvard Medical School tional analysis of miRNAs; in several cases, the loss of Boston, Massachusetts 02115 miRNA-mediated target degradation has been shown 4 Computational Biology Center to cause severe yet specific morphological or physio-Memorial Sloan Kettering Cancer Center logical defects (Chen, 2004; Palatnik et al., 2003). New York, New York 10021 In animals, the situation is more complex. Because of the lack of strict sequence complementarity, miRNA targets in the transcriptomes of worms, flies, and hu-Summary mans are more difficult to detect, and miRNA-mediated regulation is likely to be more biased toward transla-MicroRNAs are small noncoding RNAs that control tional inhibition than mRNA degradation. Insight into gene function posttranscriptionally through mRNA the biological role of miRNAs in animals comes princidegradation or translational inhibition. Much has pally from genetics and is limited to few examples, inbeen learned about the processing and mechanism cluding the control of developmental timing and cellof action of microRNAs, but little is known about their fate decisions in worm (Chang et al., 2004; Johnston biological function. Here, we demonstrate that injecand Hobert, 2003; Lee et al., 1993; Lin et al., 2003; Reintion of 2O-methyl antisense oligoribonucleotides hart et al., 2000; Wightman et al., 1993), the control of into early Drosophila embryos leads to specific and cell growth, apoptosis and fat storage in Drosophila efficient depletion of microRNAs and thus permits postembryonic development (Brennecke et al., 2003; systematic loss-of-function analysis in vivo. Twenty-Hipfner et al., 2002; Xu et al., 2003), and the regulation five of the forty-six embryonically expressed microof insulin secretion in human (Poy et al., 2004). This RNAs show readily discernible defects; pleiotropy is paucity of functional data has precluded a comprehenmoderate and family members display similar yet dissive assessment of the biological relevance of miRNAtinct phenotypes. Processes under microRNA regulamediated gene regulation in animals. tion include cellularization and patterning in the blas-Inspection of the experimentally validated miRNA tartoderm, morphogenesis, and cell survival. The largest gets and mutational analyses of known target sites microRNA family in Drosophila (miR-2/6/11/13/308) is suggest that strong complementarity with the 5# end of required for suppressing embryonic apoptosis; this the miRNA (positions 2-8) is critical for the recognition is achieved by differential posttranscriptional represof target mRNAs, while pairing at the 3# end appears sion of the proapoptotic factors hid, grim, reaper, and to be more variable (Doench and Sharp, 2004; for resickle. Our findings demonstrate that microRNAs act view see Lai [2004]). Based on these findings, computaas specific and essential regulators in a wide range tional methods have been developed to predict miRNA of developmental processes. targets in vertebrate and fly transcriptomes (Enright et al., 2003; Rajewsky and Socci, 2004; Rehmsmeier et al.,
Current Biology, 2011
Background: MicroRNAs (miRNAs) are w22 nucleotide (nt) small RNAs that control development, physiology, and pathology in animals and plants. Production of miRNAs involves the sequential processing of primary hairpin-containing RNA polymerase II transcripts by the RNase III enzymes Drosha in the nucleus and Dicer in the cytoplasm. miRNA duplexes then assemble into Argonaute proteins to form the RNA-induced silencing complex (RISC). In mature RISC, a single-stranded miRNA directs the Argonaute protein to bind partially complementary sequences, typically in the 3 0 untranslated regions of messenger RNAs, repressing their expression. Results: Here, we show that after loading into Argonaute1 (Ago1), more than a quarter of all Drosophila miRNAs undergo 3 0 end trimming by the 3 0 -to-5 0 exoribonuclease Nibbler (CG9247). Depletion of Nibbler by RNA interference (RNAi) reveals that miRNAs are frequently produced by Dicer-1 as intermediates that are longer than w22 nt. Trimming of miRNA 3 0 ends occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. In vivo, depletion of Nibbler by RNAi causes developmental defects. Conclusions: We provide a molecular explanation for the previously reported heterogeneity of miRNA 3 0 ends and propose a model in which Nibbler converts miRNAs into isoforms that are compatible with the preferred length of Ago1-bound small RNAs.
Functional Characterization of Drosophila microRNAs by a Novel in Vivo Library
Genetics, 2012
Animal microRNAs (miRNA) are implicated in the control of nearly all cellular functions. Due to high sequence redundancy within the miRNA gene pool, loss of most of these 21- to 24-bp long RNAs individually does not cause a phenotype. Thus, only very few miRNAs have been associated with clear functional roles. We constructed a transgenic UAS-miRNA library in Drosophila melanogaster that contains 180 fly miRNAs. This library circumvents the redundancy issues by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches. Demonstrating the effectiveness of our library, 78 miRNAs induced clear phenotypes. Most of these miRNAs were previously unstudied. Furthermore, we present a simple system to create GFP sensors to monitor miRNA expression and test direct functional interactions in vivo. Finally, we focus on the miR-92 family and identify a direct target gene that is responsible for the specific wing phenotype induced b...
Derivation and characterization of Dicer- and microRNA-deficient human cells
RNA, 2014
We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was una...