Regulation of RNA processing and transcription by endogenous RNAi

描述(申请人提供)：RNAi干扰在基因组监测和抗病毒防御中的作用是众所周知的。然而，最近在不同生物体中的研究表明，内源性短RNA及其辅助因子ArgAerte蛋白也调节细胞核中活跃的常染色质基因。在线虫中，与活跃转录的蛋白编码基因互补的内源性siRNAs(Endo-siRNAs)存在于与核ArgAerte蛋白CSR-1的复合体中。令人惊讶的是，这些Endo-siRNA不会导致它们的目标基因沉默。CSR-1结合的Endo-siRNAs的缺陷会导致线虫严重的发育表型，最显著的是，由于染色体分离的缺陷而导致胚胎死亡。我们最近的工作表明，这种致命性很大程度上是由于组蛋白mRNAs的3？2个末端的错误加工导致核心组蛋白蛋白的耗尽。此外，我们的全球连续测序(Gro-seq)分析显示，在CSR-1途径的活性功能丧失突变体中，转录错误调节显示CSR-1靶基因的转录全局减少，隐含和反义转录增加。内源RNAi在促进正义导向的POL II转录中的作用使人想起最近发现的基因循环对转录方向性的影响。从对酵母和哺乳动物细胞的研究中出现的新观点是，所有活性基因都采用启动子-终止子环构象，这有利于 波尔二号重新启动。重要的是，适当的共转录前-mRNA加工促进了基因环的形成，许多3？2端加工因子参与了基因环的形成。这里提出的实验旨在测试两个相关的想法：1)CSR-1途径突变体中出现的转录缺陷是由于基因环(目标1和2)的破坏，以及2)CSR-1结合的Endo-siRNAs的主要作用是促进前mRNA的加工(剪接，或3？2端的形成，或两者兼而有之)，这反过来又刺激基因环(目标3)。此外，由于缺乏Gro-Seq分析证实基因环对促进有义转录的全球贡献，我们建议分析基因环形成所需的保守因子的突变株中的转录(目标2)，并建立线虫作为适合于进一步研究RNA加工、转录和染色质结构之间关系的遗传、分子和基因组学生物。这项拟议的研究将为内源RNAi在线虫活性基因的全球调控中的积极作用提供新的见解，这可能是后生动物基因调控的重要组成部分。

DESCRIPTION (provided by applicant): The role of RNAi interference in genome surveillance and antiviral defense is well known. However, recent studies in diverse organisms suggest that endogenous short RNAs and their co-factor Argonaute proteins also regulate active euchromatic genes in the nucleus. In C. elegans, endogenous siRNAs (endo-siRNAs) complementary to actively transcribed protein-coding genes exist in a complex with the nuclear Argonaute protein CSR-1. Surprisingly, these endo-siRNAs do not cause silencing of their target genes. Deficiencies in CSR-1-bound endo-siRNAs cause severe developmental phenotypes in C. elegans, most notably, embryonic lethality due to defects in chromosome segregation. Our recent work revealed that this lethality is largely due to a depletion of core histone proteins as result of misprocessing of 3¿2 ends of histone mRNAs. Moreover, our Global Run-On Sequencing (GRO-seq) analysis of transcription misregulation in viable loss-of-function mutants of the CSR-1 pathway revealed a global reduction in transcription of CSR-1 target genes and an elevation in cryptic and antisense transcription. This role of endogenous RNAi in promoting sense-oriented Pol II transcription is reminiscent of the recently discovered effects of gene looping on transcription directionality. The emerging view from studies in yeast and mammalian cells is that all active genes adopt a promoter- terminator looping conformation, which facilitates Pol II re-initiation. Importantly, proper co-transcriptional pre- mRNA processing facilitates gene loop formation and many 3¿2-end processing factors have been implicated in gene looping. The experiments proposed here are aimed at testing two related ideas: 1) that transcriptional defects seen in CSR-1 pathway mutants are due to the disruption of gene loops (Aims 1 and 2), and 2) that the primary role of CSR-1-bound endo-siRNAs is to promote pre-mRNA processing (splicing, or 3¿2-end formation, or both), which in turn stimulates gene loops (Aim 3). Moreover, because GRO-seq analyses confirming a global contribution of gene looping to the enhancement of sense-oriented transcription are lacking, we propose to analyze transcription in mutants of conserved factors required for gene loop formation (Aim 2) and to establish C. elegans as a suitable organism for further genetic, molecular and genomic studies of the connection between RNA processing, transcription and chromatin architecture. The proposed research will provide new insights about the positive role of endogenous RNAi in global regulation of active genes in C. elegans, which may represent an essential component of gene regulation in metazoans.