Regulation of RNA processing and transcription by endogenous RNAi

描述（由申请人提供）：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，最近在多种生物中的研究表明，内源性短rna及其辅助因子Argonaute蛋白也调节细胞核中活跃的常染色基因。在秀丽隐杆线虫中，与活跃转录的蛋白质编码基因互补的内源性sirna （endodo - sirna）存在于与核Argonaute蛋白CSR-1的复合体中。令人惊讶的是，这些内切sirna不会导致它们的靶基因沉默。在秀丽隐杆线虫中，缺乏csr -1结合的内切sirna会导致严重的发育表型，最显著的是由于染色体分离缺陷导致的胚胎致死。我们最近的研究表明，这种致死率主要是由于组蛋白mrna的3 - 2端加工不当导致核心组蛋白的耗竭。此外，我们的全球运行测序（GRO-seq）分析了存活的CSR-1通路功能丧失突变体的转录失调，发现CSR-1靶基因的转录整体减少，而隐式和反义转录增加。内源性RNAi在促进感觉导向的Pol II转录中的作用让人想起最近发现的基因环对转录方向性的影响。从酵母和哺乳动物细胞的研究中出现的新观点是，所有的活性基因都采用启动子-终止子环的构象，这有利于

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.