mRNA decay in Saccharomyces cerevisiae.

Abstract

mRNA decay is an important step in the control of gene expression. To study mRNA degradation I have exploited the genetic, biochemical, and molecular tools available in Saccharomyces cerevisiae. These studies provided insight into the signals within individual transcripts which specify their half-lives, the various mechanisms by which mRNAs are degraded, and the trans-acting factors which both perform and control nucleolytic events. I identified a 65 nucleotide segment from the coding region of the unstable MATɑl mRNA which was capable of targeting both the MATɑl and stable PGKI transcripts for rapid degradation. This "instability element" was divided into two parts, one located in the first 33, and the second in the latter 32, nucleotides. The first part could be functionally replaced by different mRNA sequences containing rare codons, and while unable to promote mRNA decay by itself, enhanced degradation mediated by the second part. I determined that the MATɑl Instability Element (MIE) targets mRNAs for rapid degradation by increasing the rates of two nucleolytic steps in a pathway of mRNA decay common to several stable and unstable yeast transcripts. The initial step in this pathway is shortening of the poly(A) tail of an mRNA. Subsequently, mRNAs are decapped, after which the transcript body is degraded in a 5' to 3' exonucleolytic manner. The MIE promotes decay of the MATɑl mRNA through an increase in its decapping rate. In contrast, PGKI mRNA decay was stimulated through an increase in its rate of deadenylation. The observation that the poly(A) tail must be removed prior to mRNA decapping suggests that the poly(A) tail inhibits decapping. I determined that the major poly(A)binding protein (Pablp) is required for the inhibition of decapping mediated by the poly(A) tail. Pablp is also required for normal deadenylation rates. Pablp therefore affects mRNA decapping and deadenylation, the two rate determining steps in a common pathway of mRNA decay. Determining how Pablp, and additional trans-acting factors, exert influence over both decapping and deadenylation will provide a greater understanding of the basis of differential rates of mRNA degradation.