If there are errors in pre-mRNA processing, due to incomplete or aberrant splicing, then mRNAs would be produced that will contain premature stop codons. These mRNAs would encode truncated proteins some of which might have dominant negative activity. The cell has mechanisms to rapidly destroy these mRNAs. Dr. Maquat was one of the first to describe this phenomenon in mammalian cells and has done much of the work leading to our current understanding of this phenomenon. In this application, she proposes to extend these studies to start defining the precise biochemical mechanisms which the cell uses to identify a premature stop codon, and distinguish it from a normal stop codon. She has determined that the premature stop codon must be located a minimal distance upstream of the last intron. The degradation is often occurs with mRNA that is associated with the nucleus. Experiments where suppressor tRNAs have been expressed, demonstrate that the identification of the stop codon occurs during a process which is indistinguishable from translation. These observations have led to a model where the mRNA is """"""""marked"""""""" during splicing to identify regions that should encode proteins. Any stop codons in these regions result in the mRNA being subjected to rapid decay. This """"""""mark"""""""" is removed, possibly during the first round of translation, and if the mRNA is not recognized as defective then it retains a normal half-life. Dr. Maquat's hypothesis is that during splicing a protein(s) remains associated with the exon-exon junction serving as the """"""""mark."""""""" The goals of this proposal are to test this hypothesis. Specifically she will characterize the proteins that remain associated with exon-exon junctions after splicing and their role in NMD. Three proteins have been detected by UV-crossslinking in preliminary work: Determine if the human homologues to ubf2 and Ubf3 function in NMD in human cells. The phenotype of a mutant in the C. elegans homologue of Ubf3 is similar to that of other mutants in NMD in C.elegans. Characterize the human homologue of Ubf1 and its role in NMD, in particular studying the role of its ATPase and putative helicase activities, and what cellular proteins it interacts with.
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