The role of U1 snRNP proteins in snRNP biogenesis and gene expression regulation

U1 snRNP 蛋白在 snRNP 生物发生和基因表达调控中的作用

• 批准号: 10796664
• 负责人: Byung Ran Ranny So
• 金额: $ 42.25万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-07 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796664
• 关键词: 3' Untranslated Regions; 5' Splice Site; Address; Affinity Chromatography; Alternative Splicing; Award; Base Pairing; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biogenesis; Biological Assay; Cancer Patient; Cell physiology; Cells; Characteristics; Chemistry; Code; Complex; Core Assembly; Coupled; Data; Development; Disease; Ensure; Environment; Eukaryota; Formaldehyde; Gatekeeping; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; Grant; Immunoprecipitation; In Vitro; Introns; Length; Link; Malignant Neoplasms; Maps; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Chaperones; Motor Neurons; Mutation; Oncogenes; Outcome; Play; Poly A; Polyadenylation; Productivity; Protein Isoforms; Proteins; Quality Control; RNA; RNA 3' End Processing; RNA Biochemistry; RNA Processing; RNA Sequences; RNA Splicing; RNA metabolism; RNA-Binding Proteins; Regulation; Reporter; Reporter Genes; Research; Ribonucleoproteins; Role; SMN protein (spinal muscular atrophy); Signal Transduction; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Spliceosomes; System; Training; Transcript; Tumor Suppressor Proteins; U1 Small Nuclear Ribonucleoprotein; U1 small nuclear RNA; U1A protein; Untranslated RNA; Work; cancer cell; career; crosslink; interdisciplinary approach; knock-down; leukemia; medulloblastoma; member; novel; particle; premature; prevent; protein complex; recruit; stem; termination factor; tumor; undergraduate student

PROJECT SUMMARY The long-term goal of this proposal is to understand the regulatory mechanisms of U1 small nuclear ribonucleoprotein (U1 snRNP) in snRNP biogenesis and its role in protein-coding gene regulation. U1 snRNP, the most abundant RNP particle among the spliceosomal snRNP, plays a key role in excising introns (splicing) and preventing premature termination of nascent transcripts (telescripting). Both activities are ensured by the RNA:RNA base-pairing between U1 snRNA and 5’ splice site (5’ss). Pan-cancer patients possess multiple mutations in the U1 snRNA, including 5’ss and U1 snRNP-specific protein binding sequences, which result in alternative splicing of oncogenes and tumor suppressors through novel 5’ss recognition. However, our preliminary data showed that those U1 snRNA mutations do not form a stable Sm core, a key intermediate assembled by the SMN (survival of motor neuron) complex, which determines the stability and abundance of the U1 snRNP in cells. Moreover, we identified that a U1 snRNP-specific U1C protein, previously known to stabilize 5’ss:U1 snRNA base-pairing, plays a critical role in regulating Sm core assembly of all snRNAs. The objective of this proposal is to investigate the mechanism of the newly discovered role of U1C as a gatekeeper in snRNP biogenesis, its potential contribution to the quality control of spliceosomes, and the regulatory mechanism in splicing and telescripting activity. This project will simultaneously address both functions of U1C by harnessing two established components to dissect the role of U1C in snRNP biogenesis as well as its involvement in intronic polyadenylation and 3’UTR length changes, molecular characteristics of the oncogenes in cancer. We propose to pursue three specific aims: 1) Elucidate the role of U1C with SMN complex as a gatekeeper in snRNP biogenesis. 2) Investigate the effects of U1 snRNA mutations found in pan-cancer patients on Sm core assembly. 3) Examine the U1C interaction with mRNA 3’-end termination machinery in telescripting. Using biochemical approaches, we will investigate the molecular connections between U1C-SMN complex and U1C-U1 snRNA. By examining these connections, we can better understand how U1C controls the snRNP repertoire and ultimately impact the function of spliceosome. Moreover, we will delineate the U1C’s interaction with mRNA 3’- end processing machinery. This interaction is a key switch that can convert U1 snRNP from productive splicing to premature mRNA termination or 3’UTR shortening through the loss of 5’ss binding. To elucidate the multi- faceted regulation mechanism of U1C, we will employ in vitro Sm core assembly, RNA-affinity purification, in vitro mRNA processing assays, and in-cell formaldehyde-crosslinking coupled with immunoprecipitation methods. The expected outcome of this project will identify the role of U1C as a in snRNP biogenesis and mRNA metabolism, thus explaining the consequence of the U1 snRNA mutations in pan-cancer and providing evidence of gene regulation by the loss of telescripting activity.

项目摘要 本提案的长期目标是了解U1小型核反应堆的监管机制， 核糖核蛋白（U1 snRNP）在snRNP生物发生及其在蛋白质编码基因调控中的作用。U1 snRNP， 剪接体snRNP中最丰富的RNP颗粒，在切除内含子（剪接）中起关键作用 以及防止新生转录物的过早终止（转录终止）。这两项活动都由 RNA：U1 snRNA和5'剪接位点（5' ss）之间的RNA碱基配对。泛癌症患者具有多种 U1 snRNA中的突变，包括5 'ss和U1 snRNP特异性蛋白结合序列，其导致 癌基因和肿瘤抑制基因通过新的5 'ss识别的选择性剪接。但我们的 初步数据显示，这些U1 snRNA突变不能形成稳定的Sm核心， 由SMN（运动神经元存活）复合体组装，这决定了运动神经元的稳定性和丰度。 细胞中的U1 snRNP。此外，我们发现了一种U1 snRNP特异性U1 C蛋白，以前已知它可以稳定 5 'ss：U1 snRNA碱基配对在调节所有snRNA的Sm核心组装中起关键作用。客观 这项建议的目的是研究新发现的U1 C在snRNP中作为看门人的作用机制 生物发生，其对剪接体质量控制的潜在贡献，以及剪接体的调节机制， 剪接和转录活性。该项目将同时解决U1 C的两个功能， 两个已建立的组件来剖析U1 C在snRNP生物发生中的作用以及其参与内含子 多聚腺苷酸化和3 'UTR长度变化，癌症中癌基因的分子特征。我们提出 具体目标有三个：1）阐明U1 C与SMN复合体在snRNP中作为看门人的作用 生物起源。2)研究泛癌患者中发现的U1 snRNA突变对Sm核心组装的影响。 3)在转录过程中检查U1 C与mRNA 3 '端终止机制的相互作用。使用生化 我们将研究U1 C-SMN复合物和U1 C-U1 snRNA之间的分子联系。 通过检查这些连接，我们可以更好地理解U1 C如何控制snRNP库， 最终影响剪接体的功能。此外，我们还将描述U1 C与mRNA 3 '端的相互作用。 端部加工机械这种相互作用是一个关键开关，可以将U1 snRNP从生产性剪接转化为 导致mRNA提前终止或3 'UTR缩短，这是由于5' ss结合丧失所致。为了阐明多- U1 C的多方面调控机制，我们将采用体外Sm核心组装，RNA亲和纯化， 体外mRNA加工测定和细胞内甲醛去交联结合免疫沉淀 方法.该项目的预期结果将确定U1 C作为snRNP生物发生和mRNA表达中的作用。 代谢，从而解释了泛癌中U1 snRNA突变的后果，并提供证据 基因调控的丧失。