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 experimental procedures that can be used to determine the mechanism of degradation 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 regulatable promoters to perform transcriptional pulse-chase experiments, thereby allowing the determination of precursor-product relationships during the mRNA decay process. Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRNA turnover pathways. Most importantly, the combination of these three approaches can often clearly define the mechanism(s) by which a given transcript is degraded.
Copyright 1999 Academic Press.