Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila (original) (raw)
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...
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.
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...
The presence of extracellular microRNAs in the media of cultured Drosophila cells
Scientific Reports, 2018
While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals.
Genome …, 2011
Since the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5′ termini of certain miRNAs, frequent 3′ untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.
New MicroRNAs in Drosophila—Birth, Death and Cycles of Adaptive Evolution
PLoS Genetics, 2014
The origin and evolution of new microRNAs (miRNAs) is important because they can impact the transcriptome broadly. As miRNAs can potentially emerge constantly and rapidly, their rates of birth and evolution have been extensively debated. However, most new miRNAs identified appear not to be biologically significant. After an extensive search, we identified 12 new miRNAs that emerged de novo in Drosophila melanogaster in the last 4 million years (Myrs) and have been evolving adaptively. Unexpectedly, even though they are adaptively evolving at birth, more than 94% of such new miRNAs disappear over time. They provide selective advantages, but only for a transient evolutionary period. After 30 Myrs, all surviving miRNAs make the transition from the adaptive phase of rapid evolution to the conservative phase of slow evolution, apparently becoming integrated into the transcriptional network. During this transition, the expression shifts from being tissue-specific, predominantly in testes and larval brain/gonads/imaginal discs, to a broader distribution in many other tissues. Interestingly, a measurable fraction (20-30%) of these conservatively evolving miRNAs experience ''evolutionary rejuvenation'' and begin to evolve rapidly again. These rejuvenated miRNAs then start another cycle of adaptiveconservative evolution. In conclusion, the selective advantages driving evolution of miRNAs are themselves evolving, and sometimes changing direction, which highlights the regulatory roles of miRNAs.