Regulation of RNA processing and transcription by endogenous RNAi

内源性 RNAi 对 RNA 加工和转录的调节

• 批准号: 8757055
• 负责人: Alla Grishok
• 金额: $ 30.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-10 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8757055
• 关键词: Adopted; Affect; Animals; Antiviral Agents; Architecture; Binding; Biogenesis; Caenorhabditis elegans; Cell Nucleus; Chromatin; Chromosome Segregation; Chromosomes; Code; Complex; DNA Polymerase II; Defect; Development; Embryo; Eukaryota; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Transcription; Genetic Translation; Genome; Genomics; Histones; Link; Mammalian Cell; Mammals; Maps; Messenger RNA; Methods; Modeling; Molecular Conformation; Molecular Genetics; Nuclear; Nuclear Proteins; Organism; Pathway interactions; Phenotype; Play; Process; Processed Genes; Proteins; RNA; RNA Interference; RNA Polymerase II; RNA Processing; RNA Splicing; Regulation; Research; Role; Running; Sequence Analysis; Site; System; Testing; Transcription Factor TFIIB; Transcription Process; Work; Yeasts; genome-wide; insight; loss of function; mRNA Precursor; mutant; novel; promoter; public health relevance; research study; transcription factor; transcription termination

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.

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，最近在不同生物体中的研究表明，内源性短RNA及其辅因子Argonaute蛋白也调节细胞核中的活性常染色质基因。In C.在线虫中，与活跃转录的蛋白质编码基因互补的内源性siRNA（endo-siRNA）存在于与核Argonaute蛋白CSR-1的复合物中。令人惊讶的是，这些endo-siRNA不会导致其靶基因的沉默。CSR-1结合的endo-siRNA缺陷导致C.线虫，最值得注意的是，由于染色体分离缺陷的胚胎致死性。我们最近的工作表明，这种致死性在很大程度上是由于组蛋白mRNA的3 - 2端的错误加工导致的核心组蛋白蛋白的耗尽。此外，我们对CSR-1途径的可行功能丧失突变体中的转录失调进行的全局运行测序（GR 0-seq）分析揭示了CSR-1靶基因转录的全局减少以及隐蔽和反义转录的升高。内源性RNAi在促进有义Pol II转录中的作用使人想起最近发现的基因成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观点是，所有的活性基因都采用启动子-终止子环状构象，这有助于 Pol II重新启动。重要的是，适当的共转录前mRNA加工促进基因环的形成，许多3 - 2端加工因子与基因环有关。本文提出的实验旨在测试两个相关的想法：1）在CSR-1途径突变体中观察到的转录缺陷是由于基因环的破坏（目的1和2），以及2）CSR-1结合的endo-siRNA的主要作用是促进前mRNA加工（剪接，或3 - 2端形成，或两者兼而有之），这反过来又刺激基因环（目的3）。此外，由于缺乏确认基因环对有义转录增强的全局贡献的GRO-seq分析，我们建议分析基因环形成所需的保守因子的突变体中的转录（目的2），并建立C.线虫作为一个合适的生物体，进一步遗传，分子和基因组研究之间的联系RNA加工，转录和染色质结构。这项研究将为内源性RNAi在C. elegans，这可能是后生动物基因调控的重要组成部分。