CHARACTERIZATION OF CIS-ACTING SEQUENCES AND DECAY INTERMEDIATES INVOLVED IN NONSENSE-MEDIATED MESSENGER-RNA TURNOVER

Several lines of evidence indicate that the processes of mRNA turnover and translation are intimately linked and that understanding this rel ationship is critical to elucidating the mechanism of mRNA decay. One clear example of this relationship is the observation that nonsense mu tations can accelerate the decay of mRNAs in a process that we term no nsense-mediated mRNA decay. The experiments described here demonstrate that in the yeast Saccharomyces cerevisiae premature translational te rmination within the initial two-thirds of the PGK1 coding region acce lerates decay of that transcript regardless of which of the stop codon s is used. Nonsense mutations within the last quarter of the coding re gion have no effect on PGK1 mRNA decay. The sequences required for non sense-mediated mRNA decay include a termination codon and specific seq uences 3' to the nonsense mutation. Translation of two-thirds of the P GK1 coding region inactivates the nonsense-mediated mRNA decay pathway . This observation explains why carboxyl-terminal nonsense mutations a re resistant to accelerated decay. Characterization of the decay of no nsense-containing HIS4 transcripts yielded results mirroring those des cribed 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' termin us of the nonsense-containing transcript and is followed by 5'-->3' ex onucleolytic digestion. A model for nonsense-mediated mRNA decay based on these results is discussed.