MRNA TURNOVER BY ELEMENTS IN PROTEIN CODING REGION

RNA周转是一个相对未被探索的基因调控领域，特别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件对RNA衰变的调控机制，称为CRDIs(Coding Region Definants Of Installation)。C-fos CRDIs介导的RNA衰变与正在进行的翻译密切相关，需要核糖体转运。因此，该机制为实现对细胞生长和分化过程中基因瞬时表达的严格控制提供了极其强大的手段。此外，c-fos基因是调控细胞生长、细胞分化、应激反应和免疫反应的一大类早期反应基因，包括转录因子、原癌蛋白、细胞因子和生长因子的编码基因。一些观察表明，来自其他早期反应基因的不稳定信息也被涉及其功能由翻译触发的开放阅读框架元件的机制所降解。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个核苷酸的中心区。紫外光交联实验已经确定了几种与CRDI-1特异相互作用的蛋白质因子。目的鉴定和鉴定介导和/或调节c-fos CRDI-1失稳功能的细胞蛋白，包括CRDI结合蛋白的RNA亲和纯化、克隆相应的cDNAs和研究CRDI结合蛋白的功能作用。目的II将阐明CRDI-1对其功能和蛋白质识别所必需的关键顺式作用特征。目的III将检验基于我们的机制模型的几个预测，以解释CRDI-1介导的mRNA衰退与翻译过程中核糖体运输的耦合。目的研究哺乳动物早期反应基因mRNAs中蛋白质编码区的不稳定决定因素所介导的mRNA衰变的普遍性。本文提出的研究将为揭示基因翻译与翻译之间的耦合机制提供重要的新见解，并将揭示在细胞质基因翻译水平上实现对早期反应基因表达的严格控制的新机制(S)。此外，结果将开始描述参与原癌基因mRNA降解的蛋白质。它们可能作为癌症抑制基因发挥作用，其突变可能导致原癌基因表达失控和随后的细胞转化。

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.