Translational control of mRNA turnover in Saccharomyces cerevisiae

Abstract

Regulation of mRNA stability and mRNA translation are important components of gene expression within the eukaryotic cell. Multiple observations have suggested that the processes of translation and mRNA turnover are interrelated. Based on these observations, and the fact that the translation initiation machinery and the decapping enzyme both utilize the cap structure as a substrate, I hypothesized that these processes might be linked due to a competition at the cap between the cap binding complex and the decapping enzyme. Since disruption of translation using translational inhibitors or insertion of strong secondary structure within the 5' UTR affects the stability of mRNAs, I asked whether mutations within the translation initiation machinery itself would have a similar effect. I found that mutations in many different translation initiation factors led to an increase in the rate of mRNA turnover within the yeast cell. It was found that when the process of translation initiation is impaired in this manner that the rates of both deadenylation and decapping are increased. These results imply that the nature of the translation initiation complex bound to the 5' end of the mRNA is a critical component in determining mRNA half-life. One of the translation initiation factors, the cap binding protein eIF4E, is a logical candidate for a protein that might compete with the decapping enzyme for the cap structure. I purified the decapping enzyme and the cap binding protein from yeast and showed that addition of eIF4E could block decapping by the Dcp1p in an in vitro decapping assay. In addition, this inhibition was dependent on eIEF4E's ability to bind to the cap structure. This observation was then recapitulated in vivo by showing that an allele of eIF4E, which is unable to bind the cap structure, could suppress the decapping defect of the partially functional dcpl-1 allele. This same allele could not suppress the decapping defects of a lsm1Delta or pat1Delta, other mutations which affect decapping. These results argue that the translation initiation machinery acts as a physical block to the decapping enzyme and that decapping is composed of at least two genetically separable steps.