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 该提议的长期目标是了解U1小核的调节机制 SNRNP生物发生及其在蛋白质编码基因调节中的作用中的核糖核蛋白（U1 SNRNP）。 U1 snrnp， 剪接体SNRNP中最丰富的RNP粒子在切除介绍（剪接）中起关键作用 并防止过早终止新生转录本（伸缩）。两项活动均由 RNA：U1 snRNA和5'剪接位点（5'SS）之间的RNA碱基对。 Pan-Cancer患者潜力多重 U1 snRNA中的突变，包括5’S和U1 SNRNP特异性蛋白结合序列，这导致 通过新颖的5’s识别，癌基因和肿瘤补充剂的替代剪接。但是，我们的 初步数据表明，这些U1 snRNA突变不形成稳定的SM核，一个关键中间体 由SMN（运动神经元的生存）组装而成，该复合物决定了稳定性和抽象 细胞中的U1 SNRNP。此外，我们确定了U1 SNRNP特异性U1C蛋白，以前已知可以稳定 5'SS：U1 snRNA碱基对，在调节所有SNRNA的SM核心组件中起着至关重要的作用。目标 该建议的是研究U1C作为SNRNP中新发现的作用的机理 生物发生，其对剪接体的质量控制的潜在贡献以及调节机制 剪接和伸缩活动。该项目将通过利用U1C的两个功能来解决 两个建立的成分，用于剖析U1C在SNRNP生物发生中的作用及其参与内含子的作用 聚腺苷酸化和3'UTR的长度变化，癌症中癌基因的分子特征。我们建议 购买三个具体目标：1）阐明用SMN复合物作为守门人在SNRNP中的角色 生物发生。 2）研究Pan-Cancer患者在SM核心组件中发现的U1 SNRNA突变的影响。 3）检查伸缩伸缩的mRNA 3'-end终止机械的U1C相互作用。使用生化 方法是，我们将研究U1C-SMN复合物与U1C-U1 snRNA之间的分子连接。 通过检查这些连接，我们可以更好地了解U1C如何控制SNRNP曲目和 U1C与mRNA的互动3'-- 结束处理机械。这种交互是一个钥匙开关，可以从生产剪接中转换U1 SNRNP 过早的mRNA终止或3'UTR缩短，通过损失5’s绑定。阐明多 U1C的刻面调节机制，我们将在体外SM核心组件，RNA亲和力纯化中使用 体外mRNA加工测定法和甲醛 - 甲醛 - 连接和免疫沉淀 方法。该项目的预期结果将确定U1C在SNRNP生物发生和mRNA中的作用 代谢，从而解释了泛伴侣中U1 snRNA突变的后果，并提供了证据 通过伸缩活性的丧失来调节基因调节。