Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA.
Nature. 2010 Jun 24;465(7301):1097-101. doi: 10.1038/nature09095. Epub 2010 Jun 13.
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei. Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3' to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3' to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.
真核细胞表达多种内源性小分子调节 RNA,这些小分子调节 RNA 调节异染色质形成、发育时间、防御寄生核酸和基因组重排。许多小分子调节 RNA 被认为在核内发挥作用。例如,在植物和真菌中,短干扰 RNA(siRNA)与新生转录本结合,并指导染色质和/或 DNA 修饰。为了进一步了解小分子调节 RNA 的生物学作用,我们进行了一项遗传筛选,以鉴定秀丽隐杆线虫核中 RNA 干扰(RNAi)所需的因素。在这里,我们表明基因核 RNAi 缺陷-2(nrde-2)编码一种进化上保守的蛋白质,该蛋白质是 siRNA 介导的核内沉默所必需的。NRDE-2 在核内与 Argonaute 蛋白 NRDE-3 结合,并被 NRDE-3/siRNA 复合物招募到已被 RNAi 靶向的新生转录本上。我们发现,核定位的 siRNA 指导 RNAi 靶点 3'端的前信使 RNA(pre-mRNA)的 NRDE-2 依赖性沉默、RNA 聚合酶(RNAP)II 在被 RNAi 靶向的基因组位点的 NRDE-2 依赖性积累,以及 RNAi 靶点 3'端的 NRDE-2 依赖性 RNAP II 占据和 RNAP II 转录活性降低。这些结果将 NRDE-2 定义为核 RNAi 机制的一个组成部分,并表明后生动物 siRNA 可以共转录沉默核定位的 RNA。此外,这些结果建立了一种新的 RNAP II 调节模式:siRNA 指导 NRDE 因子的募集,该因子在转录延伸阶段抑制 RNAP II。
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