Analyzing mRNA decay in Saccharomyces cerevisiae.

Publisher Summary This chapter discusses the process of mRNA decay in Saccharomyces cerevisiae ( S. cerevisiae ) and describes the experimental procedures that can be used to determine the rates of mRNA turnover in yeast, as well as the specific pathway of degradation for any given transcript. The methods described in this chapter are used routinely to examine cytoplasmic mRNA decay events and can also be used to monitor nuclear mRNA turnover. In more complex eukaryotes, mRNA turnover can also be initiated by specific endonucleolytic cleavage. It is anticipated, but not yet observed, that some yeast mRNAs are targeted by specific endonucleases. Defects in mRNA metabolism may be a secondary result from a defect in other cellular processes. This possibility should be considered while analyzing new factors believed to alter mRNA turnover. The chapter presents a protocol of mRNA synthesis inhibition, either in general or for specific genes, and, at various times after such inhibition, the abundance of particular mRNAs is monitored by simple techniques.