Abstract
microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22–24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.
Highlights
MiRNAs are a class of ~22–24 nt endogenous regulatory RNAs present in all cell types of multicellular organisms [1,2]
Mature miRNAs are incorporated into the miRNA-induced silencing complexes (miRISC) effector complexes that contain AGO family member proteins and serve as specificity determinants to guide miRISCs to their target RNAs
Our analysis reveals that defects in the miRNA pathway can be uncoupled from those in messenger RNA processing, and that the miRNA biogenesis and messenger RNA processing machineries are physically and functionally distinct entities
Summary
MiRNAs are a class of ~22–24 nt endogenous regulatory RNAs present in all cell types of multicellular organisms [1,2]. By regulating the expression of diverse target RNAs, miRNAs play a key role in myriad biological processes including development, homeostasis, and innate immunity. In Drosophila, canonical miRNA biogenesis starts with RNA polymerase II-mediated transcription of long stem-loop primary miRNA transcripts (pri-miRNAs). They are processed in the nucleus by the Drosha/Pasha ribonuclease III (RNase III) microprocessor complex into ~60–70 nt precursor miRNAs (pre-miRNAs), via a reaction referred to as cropping [3,4,5]. Precursor miRNAs are subsequently exported to the cytoplasm by Exportin5/Ran-GTP and further processed in a dicing reaction by a second RNase III complex, the Dicer 1 (Dcr-1)/ Loqs-PB complex, thereby liberating ~22–24 nt miRNA duplexes [6,7,8,9,10,11,12]. The mature miRNA strand of the duplex is predominantly incorporated into Argonaute 1 (AGO1)containing miRNA-induced silencing complexes (miRISC). miRISC in turn engages target mRNAs via complementary base pairing between the seed region of miRNAs (positions 2–8) and miRNA-binding sites (primarily in the 3’ UTR of target mRNAs), and represses gene expression post-transcriptionally by promoting target mRNA destabilization and/or translation inhibition [13,14,15,16,17,18]
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