A unique feature of T-cell receptor (TCR) and immunoglobulin (Ig) genes is that they undergo rearrangements during lymphocyte development. A consequence of these programmed mutational events is that approximately two thirds of the rearranged TCR and Ig genes are out of frame, and thus contain premature termination codons (PTCs). Studies have demonstrated that PTCs cause TCR and Ig mRNA levels to be dramatically downregulated. This process, termed nonsense codon-mediated downregulation (NMD), has been shown to depress of expression of deleterious truncated proteins, and thus it may be mediated by a surveillance mechanism that protects cells from dominant negative-mutant proteins. Interestingly, TCR and Ig transcripts are more strongly downregulated by the NMD mechanism than are other known mammalian transcripts, suggesting the possibility that rearranging genes which frequently acquire PTCs have evolved mechanisms to amplify the NMD response. Because nonsense codons are only known to be recognized by the cytoplasmic translational machinery, it was expected that NMD would occur in the cytoplasm. Unexpectedly, several lines of evidence suggest that NMD occurs in the nucleus of lymphoid cells, including the fact that an intron is required downstream of a nonsense codon to engage the downregulatory response. Further evidence for a role of the nucleus is the finding that nonsense codons increase the levels of alternatively spliced TCR transcripts that have excised the offending PTC.
The specific aims of this application are: (1) To explain the paradoxical effects of nonsense codons on nuclear-associated events. (2) To assess whether nonsense codon involves a translation- like mechanism (although a codon-induced event would normally be assumed to require translation, several lines of recent evidence suggest this might not be the case). (3) To identify the factors responsible for the robust downregulation of TCR and Ig mRNAs in response to PTCs. (4) To elucidate whether TCR and Ig mRNAs harboring PTCs are downregulated by a common mechanism that differs from that which acts on other mammalian transcripts. The study of NMD will contribute significantly to our understanding of the immune system. Elucidation of how nonsense codons regulate nuclear events may alter prevailing views of gene expression and its compartmentalization in eukaryotic cells.
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