Polysome-associated mRNAs are substrates for the nonsense-mediated mRNA decay pathway in Saccharomyces cerevisiae

Abstract

In eukaryotic cells, premature termination of translation at nonsense codons has been implicated as the cause of a variety of posttranscriptional events, including rapid mRNA decay in the cytoplasm or the nucleus, altered splice site selection, and exon skipping. In the yeast Saccharomyces cerevisiae, nonsense codons promote accelerated mRNA decay, and we sought to determine the cellular location in which this degradation occurs. In this report, we demonstrate that six different mRNAs, including nonsense-containing transcripts of the LEU2, HIS4, PGK1, and CYH2 genes, and two wild-type mRNAs (the MAT(alpha)1 and CYH2 mRNAs), were stabilized when the translation elongation inhibitor cycloheximide was added to cellular growth media. Subsequent removal of cycloheximide resulted in resumption of translation and degradation of wild-type and nonsense-containing mRNAs. A significant fraction of the CYH2 pre-mRNA that accumulated in the presence of cycloheximide was associated with polysomes, but disappeared from that fraction when decay resumed in the absence of the drug. Moreover, the abundance of the spliced and unspliced forms of the untranslated U3 snRNA was shown to be unaffected in strains harboring mutations that stabilize nonsense-containing mRNAs. Taken together, these observations indicate that nonsense-containing mRNAs in yeast are degraded within the polysome compartment of the cell.