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: 10726886
• 负责人: Daniel Louis Kiss
• 金额: $ 11.37万
• 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/10726886
• 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_项目概要/摘要 N7-甲基鸟苷（m7 G）帽是一种独特的分子标识符，是转录后调控的焦点。 基因调控途径m7 G帽既是降解mRNA的酶的障碍， 和帽结合蛋白的着陆垫，所述帽结合蛋白协调前体mRNA加工、核输出， 大多数mRNA的翻译起始。直到最近，封顶还被认为完全是核武器， 人们认为去帽会不可逆地使RNA降解。简单地说，细胞质加帽是 m7 G帽在细胞质中返回到未加帽的mRNA的过程。胞质帽 需要NCK 1协调未知激酶、加帽酶和RNA的顺序作用 甲基转移酶，其磷酸化并加帽靶mRNA，并甲基化新添加的帽 分别虽然我们已经了解了很多关于细胞质封盖的生物化学，但许多基本的生物学原理仍然存在。 问题仍然没有答案。驱动这一提议的假设是：（1）特定的RNA序列 元件（或修饰）募集和/或触发细胞质加帽活性，以及（2）细胞质加帽活性 5 '-截短的mRNA的加帽作为转录后基因调控的新层次。本研究将寻求 三个关键问题的答案。首先，数据挖掘和新测序实验的结合将 揭示将mRNA靶向细胞质帽机制的序列。生物信息学方法 整合标记帽位置和转录起始位点（TSS）的公开数据集将识别非TSS- 相关的Caps牛津纳米孔直接RNA测序将比较从细胞+/- 显性负性细胞质加帽组分以绘制全长mRNA序列。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， 细胞质加帽的mRNA。第二，mRNA去帽酶（Dcp 2、DcpS等）的CRISPR敲除 并且候选核酸内切酶将鉴定产生未加帽末端的细胞机制， 细胞质封盖机制。这些敲除将与聚焦和转录组范围的方法配对 来验证细胞质封盖的变化第三，结合体内RNA标记实验和 核糖体分析将确定细胞质帽在应激开始时如何测量mRNA 反应5 '-截短的mRNA的产生、细胞质加帽和翻译成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， 对N-末端截短蛋白的功能的影响。总之，这项工作将（1）确定和 验证调节细胞质帽的序列（2）确定RNA 产生用于细胞质加帽的底物，和（3）理解细胞质加帽的体内功能。 在急性应激反应开始时达到顶峰。

项目成果

CHD6 promotes broad nucleosome eviction for transcriptional activation in prostate cancer cells.

• DOI: 10.1093/nar/gkac1090
• 发表时间: 2022-11-28
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Zhao, Dongyu;Zhang, Min;Huang, Shaodong;Liu, Qi;Zhu, Sen;Li, Yanqiang;Jiang, Weihua;Kiss, Daniel L.;Cao, Qi;Zhang, Lili;Chen, Kaifu
• 通讯作者: Chen, Kaifu

Polygenic risk impacts PDGFRA mutation penetrance in non-syndromic cleft lip and palate.

多基因风险影响非综合征性唇裂和腭裂的 PDGFRA 突变外显率。

• DOI: 10.1093/hmg/ddac037
• 发表时间: 2022
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Yu,Yao;Alvarado,Rolando;Petty,LaurenE;Bohlender,RyanJ;Shaw,DouglasM;Below,JenniferE;Bejar,Nada;Ruiz,OscarE;Tandon,Bhavna;Eisenhoffer,GeorgeT;Kiss,DanielL;Huff,ChadD;Letra,Ariadne;Hecht,JacquelineT
• 通讯作者: Hecht,JacquelineT