Identifying the sequences and factors that govern the fate of elongating RNAPII

鉴定控制延长 RNAPII 命运的序列和因素

基本信息

批准号：
10534168
负责人：
Karen L Adelman
金额：
$ 41.8万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10534168
关键词：
5&apos Splice Site Address Affect Antisense RNA Architecture Automobile Driving Base Pairing Base Sequence Behavior Binding Proteins Biochemical Biogenesis Bioinformatics Biological Assay CRISPR/Cas technology Cell Line Cell-Free System Cells Chromatin Chromatin Remodeling Factor Cis-Acting Sequence Code Complement Complex DNA DNA Damage DNA Insertion Elements DNA Repair DNA biosynthesis Data Data Set Development Disease Dissection Dissociation Double-Stranded RNA Enhancers Environment Enzymes Eukaryota Evolution Exhibits Exons Fluorescence Gene Expression Generations Genes Genetic Transcription Genomic Instability Genomics Goals Growth Homeostasis Human Immune System Diseases In Vitro Introns Length Libraries Malignant Neoplasms Mammals Mass Spectrum Analysis Measures Mediating Messenger RNA Methods Modeling Molecular Monitor Mus Mutate Mutation Nature Nerve Degeneration Nucleoplasm Play Poly A Polyadenylation Polymerase Prevalence Production Productivity Property Proteins RNA RNA Polymerase II RNA Processing RNA Splicing RNA analysis RNA chemical synthesis Recombinant Proteins Regulation Regulatory Element Reporter Research Role Signal Transduction Suggestion Surface System Terminator Regions Testing Tissues Transcript Transcription Elongation Transcription Initiation Site U1 Small Nuclear Ribonucleoprotein U1 small nuclear RNA Untranslated RNA Variant Work Xenopus base chromatin modification cis acting element egg embryonic stem cell enhancing factor experimental study genomic locus in vivo innovation interest mutant novel premature prevent promoter protein complex public health relevance recruit response screening small molecule termination factor transcription termination transcriptome sequencing

项目摘要

PROJECT SUMMARY: Precise control of gene expression during development and in response to signals is essential for organismal growth and homeostasis. Accordingly, gene expression is regulated at multiple steps, with tight control over transcription elongation by RNA polymerase II (RNAPII) and coordinated processing of messenger RNA (mRNA). Recent work by our lab and others has revealed that elongating RNAPII can be targeted for premature termination. Intriguingly, across eukaryotes, mammals display an increased prevalence of premature termination and “transcription attrition” within mRNAs. This phenomenon is enriched among genes involved in signaling, the DNA damage response, development and tissue-specific functions. Although the reasons for this remain to be defined, genes in these classes are often long and harbor extensive first introns, leading to suggestions that intron expansion during evolution enabled the acquisition of cryptic termination-promoting sequences. Clearly, full-length mRNA synthesis is essential for proper protein production. Accordingly, intronic termination has emerged as a contributor to many diseases, including immune dysfunction, neurodegeneration and cancer. Here, we propose to systematically define the cis-acting sequences and trans-acting protein factors that determine the fate of the RNAPII elongation complex and nascent RNA. We will define how RNAPII elongation is regulated at mRNA loci to prevent inappropriate 3’ end formation and production of aberrant transcripts, and conversely, how transcription of enhancer and antisense RNAs is rapidly terminated to prevent generation of unwanted non-coding RNA (ncRNA) species. To accomplish these goals, we developed synergistic in vivo and in vitro systems. In Aim 1, we will use a powerful screening strategy in mouse embryonic stem cells to define the sequences and proteins that influence RNAPII elongation properties and RNA fate. To complement these cell-based approaches, Aim 2 will make use of a novel cell-free transcription system to dissect the biochemical mechanisms that control RNAPII elongation and the interplay with RNA processing and termination complexes. Aim 3 will build on our preliminary data demonstrating that transcription through the first exon-intron junction stimulates RNAPII elongation rate, to investigate in detail how the sequences and protein factors involved in splicing impact RNAPII activity. This work will answer central questions about the nature of termination-promoting sequences and the factors that govern their recognition, and will describe the interactions between elongation, splicing and termination complexes. These studies will identify the requirements for elongation of a functional mRNA and the mechanisms employed to prevent transcription attrition. In parallel, we will uncover the sequences and factors that promote early termination at enhancers and other regulatory loci to prevent polymerase collisions, double-stranded RNA formation and genome instability. By elucidating these mechanisms of RNAPII control we aim to reveal the causes of, and suggest potential treatments for, the growing list of diseases involving disruption of transcription elongation, splicing and 3’ end choice.

项目概要：在发育过程中精确控制基因表达并响应信号对于生物至关重要因此，基因表达在多个步骤中受到严格控制。 RNA 聚合酶 II (RNAPII) 的转录延伸和信使 RNA 的协调加工 (mRNA) 我们实验室和其他人最近的工作表明，延长 RNAPII 可以针对早产儿。有趣的是，在真核生物中，哺乳动物的提前终止现象更为普遍。 mRNA 中的“转录损耗”现象在参与信号传导的基因中更为丰富。 DNA 损伤反应、发育和组织特异性功能，尽管其原因仍有待进一步研究。根据定义，这些类别中的基因通常很长并且具有广泛的第一个内含子，从而导致以下建议：进化过程中的内含子扩展使得能够获得神秘的终止促进序列。全长 mRNA 合成对于正确的蛋白质生产至关重要，因此，内含子终止具有重要意义。已成为许多疾病的诱因，包括免疫功能障碍、神经退行性疾病和癌症。在这里，我们建议系统地定义顺式作用序列和反式作用蛋白因子，确定 RNAPII 延伸复合物和新生 RNA 的命运我们将定义 RNAPII 延伸的方式。在 mRNA 位点进行调节，以防止不适当的 3' 末端形成和异常转录本的产生，并且相反，如何快速终止增强子和反义 RNA 的转录以防止生成为了实现这些目标，我们开发了体内和体外协同作用的RNA。在目标 1 中，我们将在小鼠胚胎干细胞中使用强大的筛选策略来定义。影响 RNAPII 延伸特性和 RNA 命运的序列和蛋白质来补充这些。基于细胞的方法，Aim 2 将利用一种新型的无细胞转录系统来剖析生物化学控制 RNAPII 延伸以及与 RNA 加工和终止复合物相互作用的机制。目标 3 将建立在我们的初步数据之上，证明转录通过第一个外显子-内含子连接刺激RNAPII延伸率，详细研究序列和蛋白质因子如何参与这项工作将回答有关终止促进性质的核心问题。序列和控制其识别的因素，并将描述伸长之间的相互作用，这些研究将确定功能伸长的要求。 mRNA 和用于防止转录损耗的机制同时，我们将揭示序列。以及促进增强子和其他调控位点提前终止以阻止聚合酶的因素碰撞、双链 RNA 形成和基因组不稳定性通过阐明 RNAPII 的这些机制。我们的目标是揭示越来越多的涉及疾病的原因，并提出潜在的治疗方法转录延伸、剪接和 3' 末端选择的破坏。