MRNA TURNOVER BY ELEMENTS IN PROTEIN CODING REGION

RNA周转代表了一个相对未开发的基因调控领域，特别是在哺乳动物细胞中。 本研究的总体目标是研究c-fos原癌基因转录本的蛋白编码区中存在的元件（称为CRDIs（编码区不稳定性决定因素））指导RNA衰变的机制和调节。 由c-fos CRDIs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，该机制提供了在细胞生长和分化期间实现对基因瞬时表达的严格控制的极其有力的手段。 此外，c-fos基因是在控制细胞生长、细胞分化、应激反应和免疫反应中具有功能的一大组早期反应基因之一，包括编码转录因子、原癌蛋白、细胞因子和生长因子的基因。 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元件的机制降解，这些元件的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的中心区域。 UV交联实验已经鉴定了几种与CRDI-1特异性相互作用的蛋白质因子。 目的鉴定和表征介导和/或调节c-fos CRDI-1去稳定功能的细胞蛋白，包括CRDI结合蛋白的RNA亲和纯化，克隆相应的cDNA，以及CRDI结合蛋白的功能作用的检查。目的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。 目的III将测试基于我们的机制模型的几个预测，以解释在翻译过程中CRDI-1介导的mRNA衰变与核糖体转运的耦合。 目的四将解决的一般性mRNA衰变介导的不稳定决定簇在哺乳动物早期反应基因mRNA的蛋白质编码区。 本文提出的研究将提供重要的新见解的机制，耦合mRNA周转翻译，并将揭示新的机制（S），通过该机制，在细胞质mRNA周转的水平上实现对早期反应基因表达的严格控制。此外，这些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因子发挥作用，其突变可导致原癌基因表达失调和随后的细胞转化。

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.