Understanding the mechanisms that regulate cytoplasmic capping and defining its contributions to post-transcriptional gene regulation

了解调节细胞质加帽的机制并定义其对转录后基因调节的贡献

• 批准号: 10655313
• 负责人: Daniel Louis Kiss
• 金额: $ 40.38万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655313
• 关键词: Alternative Splicing; Automobile Driving; Biochemistry; Bioinformatics; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoplasm; Data Set; Dominant-Negative Mutation; Elements; Ensure; Enzymes; Generations; Genes; Goals; Harvest; Knock-out; Label; Learning; Length; Maps; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; Molecular; N-terminal; Nuclear; Nuclear Export; Pattern; Phosphorylation; Phosphotransferases; Poly A; Polyadenylation; Positioning Attribute; Post-Transcriptional Regulation; Process; Protein Truncation; Proteins; Publishing; RNA; RNA Cap-Binding Proteins; RNA Processing; RNA Sequences; Regulation; Regulator Genes; Regulatory Pathway; Site; Stress; Structure; Surveys; Transcription Initiation Site; Translation Initiation; Translations; Work; acute stress; biological adaptation to stress; data mining; decapping enzyme; endonuclease; experimental study; in vivo; nanopore; posttranscriptional; recruit; ribosome profiling; transcriptome; transcriptome sequencing

R35_Project Summary/Abstract The N7-methylguanosine (m7G) cap is a unique molecular identifier that is a focal point for post-transcriptional gene regulatory pathways. The m7G cap serves as both a roadblock to enzymes that would degrade the mRNA and a landing pad for cap binding proteins that coordinate the pre-mRNA processing, nuclear export, and translation initiation of most mRNAs. Until recently, capping was thought to be exclusively nuclear, and decapping was thought to irreversibly destine the RNA to degradation. Simply stated, cytoplasmic capping is the process by which an m7G cap is returned to an uncapped mRNA in the cytoplasm. Cytoplasmic capping requires NCK1 to coordinate the sequential actions of an unknown kinase, the capping enzyme, and an RNA methyltransferase, which phosphorylate and cap the targeted mRNA and methylate the newly-added cap respectively. Although we have learned much about the biochemistry of cytoplasmic capping, many fundamental questions remain unanswered. The hypotheses driving this proposal are that: (1) Specific RNA sequence elements (or modifications) recruit and/or trigger cytoplasmic capping activity and that (2) the cytoplasmic capping of 5’-truncated mRNAs serves as a new tier of post-transcriptional gene regulation. This study will seek answers to three key questions. First, a combination of data mining and new sequencing experiments will uncover the sequences that target an mRNA to the cytoplasmic capping machinery. A bioinformatics approach integrating published data sets marking cap positions and transcription start sites (TSS) will identify non-TSS- associated caps. Oxford Nanopore direct RNA sequencing will then compare RNA harvested from cells +/- dominant negative cytoplasmic capping components to map full-length mRNA sequences. The synthesis of these studies should ascertain the 5’ ends, the alternative splicing patterns, and polyadenylation site choices of cytoplasmically capped mRNAs. Second, CRISPR knockouts of mRNA decapping enzymes (Dcp2, DcpS, etc) and candidate endonucleases will identify the cellular mechanism(s) that generate uncapped ends for the cytoplasmic capping machinery. These knockouts will be paired with focused and transcriptome-wide methods to validate changes in cytoplasmic capping. Third, a combination of in vivo RNA labeling experiments and ribosome profiling will establish how cytoplasmic capping surveys mRNAs during the onset of the stress response. The generation, cytoplasmic capping, and translation of 5’-truncated mRNAs into N-terminally- shortened proteins would effectively be a new tier of post-transcriptional gene regulation with far-reaching impacts on the function(s) of the N-terminally truncated proteins. To summarize, this work will (1) identify and validate the sequences that regulate cytoplasmic capping (2) determine the mechanism(s) by which RNA substrates are generated for cytoplasmic capping, and (3) understand the in vivo function(s) of cytoplasmic capping during the onset of acute stress responses.

R35_项目摘要/摘要 N7-甲基鸟苷(M7G)帽是一种独特的分子识别符，是转录后的焦点 基因调控途径。M7g帽既是酶的障碍，也是降解mrna的障碍。 以及一个帽子结合蛋白的着陆垫，它协调前mRNA的加工、核输出和 大多数mRNAs的翻译起始。直到最近，封顶一直被认为是完全有核的，而且 摘除被认为是不可逆转地注定了RNA的降解。简单地说，细胞质封顶是 M7G帽返回细胞质中未帽的信使核糖核酸的过程。细胞质封顶 需要NCK1来协调未知的激酶、封闭酶和RNA的顺序作用 甲基转移酶，负责磷酸化和封端靶向信使核糖核酸，甲基化新添加的封端 分别进行了分析。尽管我们已经了解了许多关于细胞质封顶的生物化学，但许多基本的 问题仍然没有得到回答。支持这一提议的假设是：(1)特定的RNA序列 元素(或修饰)招募和/或触发细胞质封顶活性，以及(2)细胞质 5‘-截短mRNAs的封顶是转录后基因调控的新层次。这项研究将寻求 三个关键问题的答案。首先，数据挖掘和新的测序实验的结合将 发现针对细胞质封顶机制的信使核糖核酸序列。一种生物信息学方法 整合已发表的标记帽位置和转录起始位点(TSS)的数据集将识别非TSS- 关联的上限。牛津纳米孔直接RNA测序将比较从细胞+/-获得的RNA 显性负性细胞质封顶成分，用于定位全长的mRNA序列。双环芳香烃的合成 这些研究应该确定5‘端、选择性剪接模式和多聚腺苷酸化位点的选择。 细胞质封顶的mRNAs。第二，mRNA解离酶(Dcp2、Dcps等)的CRISPR基因敲除 候选核酸内切酶将识别产生未封顶末端的细胞机制(S) 细胞质封顶机。这些基因敲除将与聚焦和转录组范围的方法配对。 以验证细胞质封顶的变化。第三，体内RNA标记实验和 核糖体分析将确定在应激开始时细胞质封顶如何检测mRNAs 回应。5‘-截短的mRNAs的产生、细胞质封端和翻译成N-末端- 缩短的蛋白质将有效地成为转录后基因调控的新层次，具有深远的影响 对N末端截短蛋白功能的影响(S)。总而言之，这项工作将(1)确定和 验证调节细胞质封顶的序列(2)确定核糖核酸的机制(S) 底物是为细胞质封顶而产生的，以及(3)了解细胞质的体内功能(S) 在急性应激反应开始时封顶。