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_项目总结/摘要 N7-甲基鸟苷 (m7G) 帽是一种独特的分子标识符，是转录后分析的焦点 m7G 帽既是降解 mRNA 的酶的障碍。 以及帽结合蛋白的着陆垫，协调前体 mRNA 加工、核输出和 直到最近，大多数 mRNA 的翻译起始都被认为是完全在核上进行的。 脱帽被认为会导致 RNA 不可逆地降解。简单地说，细胞质加帽是指不可逆转地导致 RNA 降解。 m7G 帽返回到细胞质中未加帽的 mRNA 的过程。 需要 NCK1 来协调未知激酶、加帽酶和 RNA 的连续作用 甲基转移酶，对目标 mRNA 进行磷酸化和加帽，并对新添加的帽进行甲基化 尽管我们已经了解了很多关于细胞质加帽的生物化学知识，但许多基本知识仍然存在。 推动这一提议的假设是：(1) 特定的 RNA 序列。 元素（或修饰）募集和/或触发细胞质加帽活性，并且（2）细胞质 5'-截短的 mRNA 的加帽可作为转录后基因调控的新层次。 首先，数据挖掘和新测序实验的结合将回答三个关键问题。 揭示将 mRNA 靶向细胞质加帽机制的序列。 整合已发布的标记帽位置和转录起始位点 (TSS) 的数据集将识别非 TSS- Oxford Nanopore 直接 RNA 测序将比较从细胞中收获的 RNA +/- 负细胞质加帽成分在绘制全长 mRNA 序列时占主导地位。 这些研究应确定 5' 末端、选择性剪接模式和聚腺苷酸化位点选择 其次，mRNA 脱帽酶（Dcp2、DcpS 等）的 CRISPR 敲除。 候选核酸内切酶将识别产生无帽末端的细胞机制 这些敲除细胞质加帽机制将与聚焦和转录组范围的方法配合使用。 第三，结合体内 RNA 标记实验和 核糖体分析将确定细胞质加帽在应激发生期间如何调查 mRNA 5'-截短的 mRNA 的生成、细胞质加帽和翻译为 N 末端。 缩短的蛋白质将有效地成为具有深远影响的新的转录后基因调控层 总而言之，这项工作将 (1) 识别和确定。 验证调节细胞质加帽的序列 (2) 确定 RNA 的机制 生成用于细胞质加帽的底物，并且（3）了解细胞质的体内功能 急性应激反应发生期间的上限。