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：
10320370
负责人：
Karen L Adelman
金额：
$ 41.83万
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/10320370
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 biosynthesis Data Data Set Development Disease Dissection 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 Location Malignant Neoplasms Mammals Mass Spectrum Analysis Measures Mediating Messenger RNA Methods Modeling Molecular Monitor Mus Mutate Mutation Nature Nerve Degeneration Play Polyadenylation Polymerase Prevalence Production 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.

项目摘要：在发育过程中和对信号的响应过程中的基因表达的精确控制对于有机体至关重要生长和体内平衡。彼此之间，基因表达受到多个步骤的调节，并严格控制 RNA聚合酶II（RNAPII）和Messenger RNA的协调处理的转录伸长（mRNA）。我们的实验室和其他人最近的工作表明，延长的rnapii可以定为目标终止。有趣的是，在真核生物中，哺乳动物表现出更高的过早终止率和mRNA中的“转录损耗”。在参与信号传导的基因中，这种现象富含 DNA损伤反应，发育和组织特异性功能。虽然这样做的原因仍然是定义，这些类别中的基因通常很长，并且藏有广泛的第一任内含子，因此提出了建议进化过程中内含子的扩展使得可以采集加密终止启动序列。清楚地，全长mRNA合成对于适当的蛋白质产生至关重要。根据内含子终止成为许多疾病的贡献者，包括免疫功能障碍，神经变性和癌症。在这里，我们建议系统地定义顺式作用序列和跨作用蛋白质因子，这些因素确定RNAPII伸长复合物和新生RNA的命运。我们将定义rnapii延长在mRNA基因座进行监管，以防止不适当的3'结束形成和异常笔录的产生，以及相反，增强子和反义RNA的转录如何迅速终止以防止产生不需要的非编码RNA（NCRNA）。为了实现这些目标，我们在体内开发了协同作用体外系统。在AIM 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伸长特性和RNA命运的序列和蛋白质。完成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化控制RNAPII伸长的机制以及与RNA处理和终止复合物的相互作用的机制。 AIM 3将基于我们的初步数据，表明通过第一个外显子 - 内部连接的转录刺激RNAPII伸长率，以详细研究如何参与的序列和蛋白质因子剪接影响RNAPII活性。这项工作将回答有关终止性质性质的中心问题序列和控制其认可的因素，并将描述伸长率之间的相互作用，剪接和终止复合物。这些研究将确定功能伸长的要求 mRNA和为防止转录流失的机制。同时，我们将发现序列以及促进增强子和其他调节区域的早期终止的因素，以防止聚合酶碰撞，双链RNA形成和基因组不稳定性。通过阐明这些RNAPII的这些机制控制我们旨在揭示涉及疾病越来越多的疾病清单的潜在治疗原因的原因转录伸长，剪接和3'最终选择的破坏。