MRNA TURNOVER BY ELEMENTS IN PROTEIN CODING REGION

点击翻译按钮获取中文摘要

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.