SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.
SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.
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DOI：
10.1371/journal.pgen.1005475
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发表时间：
2015-08
期刊：
影响因子：
4.5
通讯作者：
Zhou R
中科院分区：
文献类型：
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作者：
Xiong XP;Vogler G;Kurthkoti K;Samsonova A;Zhou R
microRNA（miRNAs）是一类内源性调控RNA，在多种生物学过程中发挥着重要作用。在转录后，初级miRNA转录物被Drosha和Dicer核糖核酸酶依次加工成~22-24 nt miRNA。随后，miRNA被掺入RNA诱导的沉默复合物（RISC）中，所述RNA诱导的沉默复合物含有Argonaute（AGO）家族蛋白，并通过互补碱基配对将RISC引导至靶RNA，从而通过翻译抑制和mRNA不稳定的组合导致转录后基因沉默。选择前体mRNA剪接因子已经涉及裂殖酵母、蠕虫、苍蝇和哺乳动物中的小RNA介导的基因沉默途径，但其潜在的分子机制还不清楚。在这里，我们表明，SmD 1，果蝇小核核糖核蛋白颗粒（snRNP）参与剪接的核心组成部分，是所需的miRNA的生物发生和功能。SmD 1与微处理器组件Pasha和pri-miRNA相互作用，并且对于最佳的miRNA生物合成是不可或缺的。SmD 1的缺失会损害miRISC的组装和功能，而不会显著影响主要经典miRNA途径组分的表达。此外，SmD 1在物理和功能上与miRISC的组成部分，包括AGO 1和GW 182相关。值得注意的是，由SmD 1沉默引起的miRNA缺陷可以与前体mRNA剪接中的缺陷解偶联，并且miRNA和剪接机制在物理上和功能上是不同的实体。最后，光活化核糖核苷增强的交联和免疫沉淀（PAR-CLIP）分析确定了许多SmD 1结合事件的转录组，并揭示了直接SmD 1-miRNA的相互作用。我们的研究表明，SmD 1在miRNA介导的基因沉默中起着直接的作用，独立于其前mRNA剪接活性，并表明剪接因子在转录后基因调控中的双重作用可能是进化上普遍存在的。microRNA（miRNAs）是一类通过减少靶信使RNA的蛋白质输出来微调基因表达的小的调节RNA，并且涉及无数的生理和病理过程。通过Drosha/Pasha和Dicer核糖核酸酶的连续作用，从长初级转录物产生miRNA。成熟的miRNAs被整合到miRISC效应复合物中，该复合物含有AGO家族成员蛋白，并作为特异性决定簇将miRISC引导至其靶RNA。以前的研究表明，选择参与信使RNA加工的蛋白质是miRNA产生/功能所必需的，但其潜在的分子机制尚未得到很好的理解。在这里，我们表明，SmD 1，一种参与信使RNA加工的必需蛋白质，直接与Pasha和初级miRNA转录本相互作用，并且是最佳miRNA产生所必需的。此外，SmD 1与miRNA效应器机制的多个组分相关，并且是miRNA功能所需的。最后，我们的分析表明，miRNA途径中的缺陷可以与信使RNA加工中的缺陷解偶联，并且miRNA生物发生和信使RNA加工机制在物理和功能上是不同的实体。因此，我们的数据表明，SmD 1调节的miRNA通路独立的信使RNA加工中的作用。
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. microRNAs (miRNAs) are a class of small regulatory RNAs that fine-tune gene expression by reducing protein output from their target messenger RNAs and are implicated in myriad physiological and pathological processes. miRNAs are generated from long primary transcripts via sequential actions of the Drosha/Pasha and Dicer ribonucleases. Mature miRNAs are incorporated into the miRISC effector complexes that contain AGO family member proteins and serve as specificity determinants to guide miRISCs to their target RNAs. Previous studies suggested that select proteins implicated in the processing of messenger RNAs are required for the miRNA production/function, but the underlying molecular mechanism is not well understood. Here we show that SmD1, an essential protein implicated in the processing of messenger RNAs, directly interacts with both Pasha and primary miRNA transcripts and is required for optimal miRNA production. Furthermore, SmD1 associates with multiple components of the miRNA effector machinery and is required for miRNA function. Finally, 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. Our data thus suggests that SmD1 modulates the miRNA pathway independent of its role in messenger RNA processing.