Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.
Department of Molecular Biology, Umeå University, 901 87, Umeå, Sweden. marcus.johansson@umu.se.
Marcus J O
MJ
Johansson
D015152:Blotting, Northern; D020869:Gene Expression Profiling / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D006207:Half-Life; D059365:Nonsense Mediated mRNA Decay; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins
Half-lives; NMD; Northern blotting; RNA isolation; Yeast; mRNA decay; mRNA levels