Relationship of cytoplasmic capping to post-transcriptional gene regulation

细胞质加帽与转录后基因调控的关系

基本信息

批准号：
9118224
负责人：
DANIEL R. SCHOENBERG
金额：
$ 32.78万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9118224
关键词：
Affect Affinity Amplifiers Binding Binding Proteins Biochemical Bioinformatics Biological Biological Assay Cell Differentiation process Cell Nucleus Cells Complement Complex Core Protein Cytoplasm Data Set Dendrites Diphosphates Drosophila genus Elements Embryonic Development Enzymes Exons Funding Gene Expression Gene Expression Profiling Gene Expression Regulation Genome Health Homeostasis In Vitro Infection Initiator Codon Internal Ribosome Entry Site Length Life Cycle Stages Location Maintenance Maps Messenger RNA Methyltransferase Mining Mitotic Cell Cycle Nature Neurons Nucleotides Outcome Paper Phase Phosphotransferases Play Positioning Attribute Post-Transcriptional Regulation Process Proline Protein Tyrosine Kinase Proteins Proteome Proteomics RNA RNA Cap-Binding Proteins RNA Sequences RNA Splicing Reporting Ribosomes Role SH3 Domains Scanning Signal Transduction Site Specificity Structure Transcript Transducers Translating Translation Initiation Translation Process Translations Vertebrates Virus Work adapter protein base beta Globin cell growth cell type in vivo insight mRNA Decay mRNA Precursor mRNA capping neuronal cell body novel strategies protein complex protein expression research study ribosome profiling src Homology Region 2 Domain transcriptome

项目摘要

DESCRIPTION (provided by applicant): This proposal deals with the unprecedented phenomenon of mRNA capping in the cytoplasm. The 5' ends of all mRNAs have an m7G `cap', and proteins that bind to the cap direct the processing, translation and fate of every transcript. The prevailing view was that caps could only be added to newly synthesized pre-mRNAs in the nucleus, and loss of the cap leads irreversibly to mRNA decay. In contrast, we identified transcripts that are stable in an uncapped state, identified a cytoplasmic complex of proteins that can restore the cap onto these transcripts, and identified a cyclical process of decapping and recapping termed `cap homeostasis' that maintains a subset of the transcriptome in an actively translating state. The process of cytoplasmic capping involves conversion of the 5'-monophosphate end of uncapped RNA to a 5'-diphosphate and the transfer of GMP from capping enzyme onto this recapping substrate. All of the enzymes needed to catalyze cytoplasmic capping are present in a single complex that assembles on Nck1, a cytoplasmic SH2/SH3 adapter protein that is best known as a transducer of tyrosine kinase signaling. The RNA 5'-kinase and capping enzyme are juxtaposed by binding to adjacent SH3 domains, and the presence of cap methyltransferase in the complex completes the list of proteins that are necessary and sufficient to affect cytoplasmic capping. Cytoplasmic capping targets are not random; they encode proteins involved in nucleotide binding, protein localization, RNA localization, and the mitotic cell cycle. The working hypothesis of this proposal is that cytoplasmic capping is a selective post-transcriptional process that functions as an amplifier of transcriptome and proteome complexity. In Aim 1 in vitro, in vivo and biochemical biological approaches will be used to characterize the 5'-kinase and its function in cytoplasmic capping. Nck1 has 4 functional domains, and classical and `top-down' proteomics and biochemical approaches will be used to identify and characterize proteins that are bound uniquely to the 1st SH3 domain and SH2 domain in the context of the cytoplasmic capping complex. Aim 2 will map the 5' ends of recapped transcripts and determine their relationship to internal cap sites identified by Capped Analysis of Gene Expression (CAGE). The resulting datasets will be mined to identify sequence and/or structural motifs that determine the location of recapped 5' ends and their role in determining target specificity. In Aim 3 ribosome profiling will be combined with positional proteomics to determine the relationship of cytoplasmic capping to translation and proteome complexity. The results will be confirmed by top-down proteomics of selected products from internally capped transcripts, and changes in subcellular distribution will be used as an assay for functional effects of downstream capping on protein diversity. In summary this work will determine the organization of the cytoplasmic capping complex, the location of recapped ends within target transcripts, and the impact of cytoplasmic capping on transcriptome and proteome complexity.

描述（由申请人提供）：该提案涉及细胞质中前所未有的 mRNA 加帽现象。所有 mRNA 的 5' 末端都有一个 m7G“帽”，与帽结合的蛋白质指导 mRNA 的加工、翻译和命运。普遍的观点是，帽子只能添加到细胞核中新合成的前 mRNA 上，帽子的丢失会导致 mRNA 不可逆地衰减，相反，我们发现了在细胞核中稳定的转录本。脱帽状态，鉴定出可以将帽恢复到这些转录物上的蛋白质的细胞质复合物，并鉴定了称为“帽稳态”的脱帽和重帽循环过程，该过程将转录组的子集维持在主动翻译状态。涉及将未加帽的 RNA 的 5'-单磷酸末端转化为 5'-二磷酸，以及将 GMP 从加帽酶转移到该重新加帽底物上。催化细胞质加帽的复合物存在于 Nck1 上组装的单个复合物中，Nck1 是一种细胞质 SH2/SH3 接头蛋白，最知名的是酪氨酸激酶信号转导器。RNA 5'-激酶和加帽酶通过与相邻的 SH3 结合而并置。结构域，并且复合物中帽甲基转移酶的存在完善了影响细胞质加帽目标所必需且充分的蛋白质列表。它们编码参与核苷酸结合、蛋白质定位、RNA 定位和有丝分裂细胞周期的蛋白质。该提议的工作假设是细胞质加帽是一种选择性转录后过程，可作为转录组和蛋白质组复杂性的放大器。在目标 1 中，将使用体外、体内和生化生物学方法来表征 5'-激酶及其在细胞质加帽中的功能，Nck1 具有 4 个功能域，以及经典和功能域。 “自上而下”的蛋白质组学和生化方法将用于识别和表征在细胞质加帽复合物的背景下与第一个 SH3 结构域和 SH2 结构域独特结合的蛋白质，目标 2 将绘制重加帽转录物的 5' 末端图谱。确定它们与通过基因表达加帽分析 (CAGE) 识别的内部帽位点的关系将挖掘所得数据集以识别确定加帽 5' 末端位置的序列和/或结构基序。在 Aim 3 中，核糖体分析将与位置蛋白质组学相结合，以确定细胞质加帽与翻译和蛋白质组复杂性的关系，结果将通过内部加帽转录本的选定产物的自上而下的蛋白质组学来证实。亚细胞分布的变化将用作下游加帽对蛋白质多样性的功能影响的测定。总之，这项工作将确定细胞质加帽复合物的组织，以及加帽末端的位置。目标转录本，以及细胞质加帽对转录组和蛋白质组复杂性的影响。