mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Abstract

Nonsense mutations in a gene can accelerate the decay rate of the mRNA transcribed from that gene, a phenomenon we describe as nonsense-mediated mRNA decay. Using amber (UAG) mutants of the yeast PGK1 gene as a model system, we find that nonsense-mediated mRNA decay is position dependent, that is, nonsense mutations within the initial two-thirds of the PGKl-coding region accelerate the decay rate of the PGK1 transcript ~<12-fold, whereas nonsense mutations within the carboxy-terminal third of the coding region have no effect on mRNA decay. Moreover, we find that this position effect reflects (1) a requirement for sequences 3' to the nonsense mutation that may be necessary for translational reinitiation or pausing, and (2) the presence of an additional sequence that, when translated, inactivates the nonsense-mediated mRNA decay pathway. This stabilizing element is positioned within the coding region such that it constitutes the boundary between nonsense mutations that do or do not affect mRNA decay. Rapid decay of PGK1 nonsense-containing transcripts is also dependent on the status of the UPF1 gene. Regardless of the position of an amber codon in the PGK1 gene, deletion of the UPF1 gene restores wild-type decay rates to nonsense-containing PGK1 transcripts.

New Methods Enabling Efficient Incorporation of Unnatural Amino Acids in Yeast

“…The yeast UPF1 gene has been shown to be essential for NMD, deletion of which restores wt decay rates to nonsensecontaining mRNA transcripts. 10 We therefore generated a upf1∆ strain of S. cereVisiae and compared the UAA incorporation efficiency in this strain to the wt strain.…”

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

“…Mutations in the UPF genes were isolated as allosuppressors of a his4 frameshift allele (17,43). Subsequent studies demonstrated that strains harboring the upf1 and upf3 alleles, and as shown in more recent studies also upf2, lead to the selective stabilization of nonsense-containing mRNAs without affecting the decay rates of most other mRNAs (16,26,42,43,56). The UPF1 and UPF2 genes have been cloned and sequenced (16,25,43).…”

“…Our results, as well as work from other laboratories, strongly indicate that the processes of mRNA turnover and translation are intimately linked and that understanding of this relationship is critical in elucidating the mechanism of mRNA decay (2, 5, 9, 11, 15, 21, 23, 29-31, 41, 53, 55-59, 76). The role of translation in determination of mRNA decay rates is direct, and, for certain instability elements identified in protein coding regions and 3Ј untranslated regions (3Ј-UTRs), translation of the protein coding region must occur in order for them to function (2,11,15,21,23,29,41,53,60,66,76).One clear example of the relationship between translation and mRNA decay is the observation that nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene in a process we term nonsense-mediated mRNA decay (55,56,58). Reduced mRNA levels or decreased stabilities of nonsense-containing transcripts have been observed in both prokaryotes and eukaryotes (6-8, 13, 14, 18, 20, 22, 42, 44-47, 51, 54, 56, 71, 75).…”

“…One clear example of the relationship between translation and mRNA decay is the observation that nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene in a process we term nonsense-mediated mRNA decay (55,56,58). Reduced mRNA levels or decreased stabilities of nonsense-containing transcripts have been observed in both prokaryotes and eukaryotes (6-8, 13, 14, 18, 20, 22, 42, 44-47, 51, 54, 56, 71, 75).…”

“…The PGK1 transcript, normally a stable and abundant mRNA, was used as our model substrate to investigate this process (24,56). Previous results have demonstrated the following (24,56): (i) nonsense codons, independent of the type, located within the amino-terminal two-thirds of the protein-coding region accelerate the decay rate of the PGK1 transcript up to 12-fold, whereas nonsense mutations located within the carboxyl-terminal one-third of the protein-coding region have no effect on decay; (ii) specific sequences 3Ј to the nonsense mutation, which we have defined as downstream elements, are required for nonsense-mediated mRNA decay; (iii) the PGK1 transcript contains a stabilizer element that, when translated, inactivates the nonsense-mediated mRNA decay pathway. The stabilizer element in the PGK1 gene is positioned within the protein-coding region such that it constitutes the boundary between nonsense mutations which do or do not affect mRNA decay.…”