Characterization of cis-Acting Sequences and Decay Intermediates Involved in Nonsense-Mediated mRNA Turnover

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Abstract
Several lines of evidence indicate that the processes of mRNA turnover and translation are intimately linked and that understanding this relationship is critical to elucidating the mechanism of mRNA decay. One clear example of this relationship is the observation that nonsense mutations can accelerate the decay of mRNAs in a process that we term nonsense-mediated mRNA decay. The experiments described here demonstrate that in the yeast Saccharomyces cerevisiae premature translational termination within the initial two-thirds of the PGK1 coding region accelerates decay of that transcript regardless of which of the stop codons is used. Nonsense mutations within the last quarter of the coding region have no effect on PGK1 mRNA decay. The sequences required for nonsense-mediated mRNA decay include a termination codon and specific sequences 3' to the nonsense mutation. Translation of two-thirds of the PGK1 coding region inactivates the nonsense-mediated mRNA decay pathway. This observation explains why carboxyl-terminal nonsense mutations are resistant to accelerated decay. Characterization of the decay of nonsense-containing HIS4 transcripts yielded results mirroring those described above, suggesting that the sequence requirements described for the PGK1 transcript are likely to be a general characteristic of this decay pathway. In addition, an analysis of the decay intermediates of nonsense-containing mRNAs indicates that nonsense-mediated mRNA decay flows through a pathway similar to that described for a class of wild-type transcripts. The initial cleavage event occurs near the 5' terminus of the nonsense-containing transcript and is followed by 5'-->3' exonucleolytic digestion. A model for nonsense-mediated mRNA decay based on these results is discussed.