Analysis of Nonsense‐Mediated mRNA Decay in Saccharomyces cerevisiae

Nonsense-mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild-type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady-state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5′ and 3′ untranslated region (UTR) lengths, and poly(A) tail length. Curr. Protoc. Cell Biol. 54:27.3.1-27.3.39. © 2012 by John Wiley & Sons, Inc. Keywords: steady-state mRNA level; mRNA decay rate; untranslated region length; 5′-cap; poly(A) tail length; nonsense suppression; allosuppression