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

• 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
• 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' 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

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体至关重要 生长和稳态。因此，基因表达受到多个步骤的调控，并严格控制 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 终止。有趣的是，在真核生物中，哺乳动物的提前终止发生率更高 以及 mRNA 内的“转录损耗”。这种现象在参与信号传导的基因中丰富， DNA 损伤反应、发育和组织特异性功能。尽管造成这种情况的原因还有待 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。清楚地， 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 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， 决定 RNAPII 延伸复合物和新生 RNA 的命运。我们将定义RNAPII如何延伸 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 不需要的非编码 RNA (ncRNA) 种类。为了实现这些目标，我们开发了体内协同作用和 体外系统。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。为了补充这些 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 剪接影响 RNAPII 活性。这项工作将回答有关终止促进的本质的核心问题 序列和控制其识别的因素，并将描述伸长之间的相互作用， 剪接和终止复合物。这些研究将确定功能延伸的要求 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。