9808936 Wilkinson Post-transcriptional events in eukaryotic cells are compartmentalized. Transcripts are spliced in the nucleus, and then translocated to the cytoplasm, where translation occurs. A surprising observation has been that premature termination codons (PTCs) affect not only cytoplasmic events but also nuclear-associated events. Some PTCs upregulate alternatively spliced (alt) mRNAs that have excised the PTC, a process called PTC-mediated upregulation (PMU). That the nucleus may be involved in PMU is further suggested by experiments demonstrating the importance of RNA splicing. The goal of this study is to understand how nonsense codons, which are only known to be read by the cytoplasmic translational machinery, regulate nuclear events. The T-cell receptor-beta (TCRBeta) gene will be used for this investigation because it commonly acquires PTCs during normal T-cell development and therefore mechanisms that monitor the acquisition of PTCs in this gene may be critical for normal immune cell function. In this project, the mechanism responsible for PMU will be studied. The objectives of the study are: (1) to elucidate the specific post-transcriptional mechanism involved (e.g., alternative splice-site selection vs. regulation of RNA stability) and (2) to experimentally evaluate cis and trans models that explain PMU. To address these issues, genetically engineered TCR constructs will be transfected into mammalian cells and the transcribed mRNAs analyzed by ribonuclease (Rnase) protection, northern blot, and reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses. A tetracycline (tet)-regulated promoter will be used for RNA half-life studies. Proteins are synthesized within the cytosol of the cell using information copied from DNA into RNA. Cellular surveillance mechanisms exist to insure that defective RNAs, which would result in the synthesis of mutant proteins, do not accumulate. This project will investigate the process by which RNAs containing mistakes ( nonsense codons) do not accumulate, while alternative forms of the mRNA, in which the mistake has been eliminated by an alternative processing event, accumulate to a higher than normal level. This investigation is important for several reasons: (1) the mechanism responsible for skipping deleterious nonsense codons by alternative processing may be physiologically important because it can permit the expression of functional proteins from otherwise defective genes, (2) this process may be a component of a general-surveillance mechanism that recognizes and destroys aberrant transcripts containing nonsense codons, and (3) an understanding of how nonsense codons affect nuclear events may alter the prevailing view of gene expression.
