This proposal addresses two aspects of the post-transcriptional regulation of gene expression in eukaryotic organisms: a) the determinants of mRNA stability and b) the cytoplasmic function of the 3'-poly(A) tracts present on mRNA. Determinants of mRNA Stability: Recent experiments have led us to conclude that random nucleolytic processes are likely to play a significant role in determining the rates of degradation of specific mRNAs. Therefore, we propose to address the possibility that specific elements (sequences or structures) of unstable Dictyostelium and yeast mRNAs are responsible for the instability of those mRNAs. To identify such instability elements we will construct: a) chimeras from genes encoding stable and unstable mRNAs and b) deletions of specific regions of genes encoding unstable mRNAs. These modified genes will be introduced into cells by DNA mediated transformation and the stability of the encoded mRNAs determined in vivo. In vitro mutagenesis procedures will subsequently be utilized to delineate the instability elements more precisely. An in vitro system will be utilized to determine whether instability elements are the targets of specific nucleases. It is anticipated that an understanding of the structural basis of the instability of specific yeast mRNAs will aid us in devising genetic selections for mutants defective in mRNA stability regulation. Cytoplasmic Function of Poly(A): Numerous indirect experiments from this lab and others suggest that poly(A) has a role in translational initiation which may be mediated by the cytoplasmic poly(A)-binding protein(s). We propose to utilize cell-free translation systems to address this hypothesis directly. We will inactivate the yeast poly(A)-binding protein, either with specific polyclonal antibodies or by using a temperature-sensitive mutant, and evaluate the effects on translation. Further, we will transcribe mRNAs with different discrete poly(A) tail lengths from modified SP6 vectors which we have constructed and determine their relative efficiencies of recruitment into polysomes. The possibility that a specific reduction in the length of the poly(A) tail on a given mRNA correlates with a shift to smaller polysomes, or with a shift off of polysomes, will also be addressed by an in vivo pulse-chase experiment in yeast.
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