MRNA TURNOVER BY ELEMENTS IN PROTEIN CODING REGION

蛋白质编码区各元素的 mRNA 周转率

• 批准号: 6386446
• 负责人: Ann-Bin Shyu
• 金额: $ 17.94万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386446
• 关键词: 3T3 cells; HeLa cells; affinity chromatography; binding proteins; cell differentiation; cell growth regulation; gene expression; genetic regulatory element; genetic translation; high performance liquid chromatography; messenger RNA; nucleic acid metabolism; polymerase chain reaction; protein degradation; protein structure function; protooncogene; regulatory gene; western blottings

RNA turnover represents a relatively unexplored area of gene regulation, especially in mammalian cells. The overall goal of this study is to investigate the mechanisms and regulation of RNA decay directed by elements present in the protein coding region of c-fos protooncogene transcript, termed CRDIs (coding region determinants of instability). RNA decay mediated by c-fos CRDIs is tightly coupled to ongoing translation and requires ribosome transit. Thus, the mechanism provides an extremely powerful means to achieve stringent control over transient expression of a gene during cell growth and differentiation. In addition, the c- fos gene is one of a large group of early-response genes which have functions in controlling cell growth, cell differentiation, stress response and immune response, including genes encoding transcriptions factors, proto-oncoproteins, cytokines, and growth factors. Several observations suggest that the labile messages from other early-response genes is also degraded by mechanisms involving open-reading-frame elements whose functions are triggered by translation. Our preliminary studies indicate that the major CRDI, termed CRDI-1, maps to a 320-nt central region of the c-fos coding region. UV cross-linking experiments have identified several protein factors that interact specifically with CRDI-1. Aim I will identify and characterize cellular proteins that mediate and/or regulate c-fos CRDI-1 destabilizing function and will include RNA-affinity purification of CRDI- binding proteins, cloning the corresponding cDNAs, and examination of the functional roles of the CRDI-binding proteins. Aim II will elucidate key cis-acting features of CRDI-1 necessary for its function and for protein recognition. Aim III will test several predictions based on our mechanistic model to explain coupling of CRDI-1 mediated mRNA decay to ribosome transit during translation. Aim IV will address the generality of mRNA decay mediated by destabilizing determinants in protein coding regions in mammalian early-response-gene mRNAs. The studies proposed here will provide important new insights into the mechanism that couples mRNA turnover to translation and will reveal novel mechanism(s) by which stringent control of early-response-gene expression is achieved at the level of cytoplasmic mRNA turnover. In addition, the results will begin characterization of proteins involved in the degradation of proto-oncogene mRNA. They may function as cancer suppressors whose mutation can lead to deregulated protooncogene expression and subsequent cell transformation.

RNA更新代表了基因调节的相对未开发的区域，尤其是在哺乳动物细胞中。 这项研究的总体目的是研究由C-Fos原子元转录本中存在的元素引导的RNA衰变的机理和调节，称为CRDIS（编码区域的不稳定性区域决定因素）。 由C-FOS CRDIS介导的RNA衰变与持续的翻译紧密耦合，需要核糖体传输。 因此，该机制提供了一种非常强大的手段，可以在细胞生长和分化过程中严格控制基因的瞬时表达。 此外，C-FOS基因是一大批早期反应基因之一，它们在控制细胞生长，细胞分化，应激反应和免疫反应方面具有起作用，包括编码转录因子，原始蛋白质，细胞因子和生长因子的基因。 几种观察结果表明，来自其他早期响应基因的不稳定消息也因涉及开放阅读框架元素的机制而降低，其功能是由翻译触发的。 我们的初步研究表明，主要的CRDI称为CRDI-1，映射到C-FOS编码区的320-NT中央区域。 紫外线交联实验已经鉴定出与CRDI-1特别相互作用的几种蛋白质因子。 目的我将识别并表征介导和/或调节C-FOS CRDI-1不稳定功能的细胞蛋白，并包括CRDININGINGING蛋白的RNA - 亲和力纯化，克隆相应的CDNA，并检查CRDI结合蛋白的功能作用。 AIM II将阐明CRDI-1功能和蛋白质识别所必需的关键顺式作用特征。 AIM III将基于我们的机械模型测试几个预测，以解释CRDI-1介导的mRNA衰变与核糖体传输过程中的耦合。 AIM IV将解决哺乳动物早期反应 - 基因mRNA中蛋白质编码区域中的决定因素介导的mRNA衰变的普遍性。 此处提出的研究将提供有关将mRNA转换与翻译的机制的重要新见解，并将揭示新的机制，通过该机制在细胞质mRNA转换水平上严格控制早期反应基因表达。另外，结果将开始表征与原始癌基因mRNA降解有关的蛋白质。 它们可能充当癌症抑制子，其突变可能导致原子量的失调和随后的细胞转化。