Analysis of mRNA decay pathways in Saccharomyces cerevisiae

The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. in this article we describe expe rimental procedures that can be used to determine the mechanism of degradat ion for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates. In addition, mRNA decay pathways can be analyzed by using re gulatable promoters to perform transcriptional pulse-chase experiments, the reby allowing the determination of precursor-product relationships during t he mRNA decay process. Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRN A turnover pathways. Most importantly, the combination of these three appro aches can often clearly define the mechanism(s) by which a given transcript is degraded. (C) 1999 Academic Press.