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: 6519992
• 负责人: Ann-Bin Shyu
• 金额: $ 20.93万
• 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/6519992
• 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 周转代表了基因调控中相对未被探索的领域，尤其是在哺乳动物细胞中。 本研究的总体目标是研究 c-fos 原癌基因转录本蛋白质编码区中存在的元素（称为 CRDI（不稳定编码区决定因素））指导的 RNA 衰变的机制和调节。 c-fos CRDI 介导的 RNA 衰变与正在进行的翻译紧密耦合，并且需要核糖体转运。 因此，该机制提供了一种极其强大的手段来实现对细胞生长和分化过程中基因瞬时表达的严格控制。 此外，c-fos基因是一大类早期反应基因之一，具有控制细胞生长、细胞分化、应激反应和免疫反应的功能，包括编码转录因子、原癌蛋白、细胞因子和生长因子的基因。因素。 一些观察结果表明，来自其他早期反应基因的不稳定信息也会被涉及开放阅读框架元件（其功能由翻译触发）的机制所降解。 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 中心区域。 UV 交联实验已鉴定出几种与 CRDI-1 特异性相互作用的蛋白质因子。 目标我将鉴定和表征介导和/或调节 c-fos CRDI-1 去稳定功能的细胞蛋白，并将包括 CRDI 结合蛋白的 RNA 亲和纯化、克隆相应的 cDNA 以及检查 CRDI- 的功能作用。结合蛋白。目标 II 将阐明 CRDI-1 的功能和蛋白质识别所必需的关键顺式作用特征。 Aim III 将测试基于我们的机制模型的多项预测，以解释翻译过程中 CRDI-1 介导的 mRNA 衰减与核糖体转运的耦合。 目标 IV 将解决哺乳动物早期反应基因 mRNA 中蛋白质编码区不稳定决定簇介导的 mRNA 衰减的普遍性。 这里提出的研究将为 mRNA 周转与翻译耦合的机制提供重要的新见解，并将揭示在细胞质 mRNA 周转水平上实现早期反应基因表达的严格控制的新机制。此外，研究结果将开始表征参与原癌基因 mRNA 降解的蛋白质。 它们可能作为癌症抑制剂发挥作用，其突变可导致原癌基因表达失调和随后的细胞转化。