UPF GENE PRODUCT FUNCTION IN TURNOVER OF NATURAL MRNAS

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UPF genes accelerate nonsense mRNA decay in Saccharomyces cerevisiae. Recent evidence indicates UPF genes directly modulate the decay of the natural CTF13 mRNA, which encodes an essential kinetochore protein. This proposal will focus on understanding the UPF-mediated decay of natural mRNA targets that code for kinetochore proteins. We aim to determine the nature of the CTF13 mRNA decay pathway, to determine to what extent UPF genes affect kinetochore mRNA levels, and to investigate the molecular mechanism of UPF-mediated decay of natural mRNAs. We will analyze the intermediates in the decay of CTF13 mRNA to determine if it is degraded by a deadenylation-dependent or -independent pathway. We will determine whether CTF13 mRNA is decapped and degraded exonucleolyticly 5'-->3' or whether it decays by another pathway. We will examine the steady-state levels and half-lives of two other kinetochore mRNAs encoded by CTF14 and CBF3b to determine if they are directly modulated by UPF function. If two or more kinetochore mRNAs are affected, we will address the possibility that UPF genes regulate the expression of kinetochore mRNAs during the cell cycle, by monitoring CTF13 mRNA accumulation in synchronized cells carrying UPF loss-of-function alleles. CTF13 and potentially other direct targets of UPF function will be studied further to define the sequences or structures in the mRNAs that allow them to be recognized as substrates of the UPF-mediated decay pathway. This will be accomplished through the analysis of target mRNAs either containing deletions or as part of lacZ gene fusions. Gel mobility shift assays will be used to address what protein(s) bind the recognition sequence(s) identified above. The long- range goal is to understand what cellular purpose is served by UPF- mediated decay of specific, natural mRNAs. As UPF-mediated mRNA decay modulates kinetochore gene expression, its study has health-related importance for cancer.