Nonsense-mediated mRNA Decay Pathway Research Articles

Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen.

Rapid turnover of nonsense-containing mRNAs in yeast in dependent on the product of the UPF1 gene (Upf1p). Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. To identify other integral components of this decay pathway, we have employed a two-hybrid screen, seeking those cellular factors that specifically interact with Upf1p. Screening of yeast genomic libraries identified six genes encoding potential Upf1p-interacting proteins. These include four previously uncharacterized genes, NMD1-4 (nonsense-mediated mRNA decay), DBP2, a gene encoding a putative RNA helicase with homology to mammalian p68 RNA helicase, and SNP1, a gene encoding a U1 snRNP 70-kD protein homolog. In this paper we report the identification and characterization of NMD2, a yeast gene that encodes a specific Upf1p-interacting protein. Disruption of NMD2 yields a nonsense-mediated mRNA decay phenotype identical to that obtained in UPF1-deletion strains, indicating that the NMD2 gene product (Nmd2p) is a new factor in the nonsense-mediated mRNA decay pathway. Deletion analysis demonstrated that the acidic carboxyl terminus of Nmd2p constituted the Upf1p-interacting domain. High-level expression of a fragment of Nmd2p containing this domain had a dominant-negative effect on nonsense-mediated mRNA decay when the protein was localized the cytoplasm but not when it was localized to the nucleus, indicating that this decay pathway has a cytoplasmic component. The association of a dominant-negative phenotype with a gene fragment identified in a two-hybrid screen suggests a generalized approach to confirming the function of genes identified in such screens.

Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover.

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.

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

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 PGK1-coding region accelerate the decay rate of the PGK1 transcript < or = 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.

Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant.

Nonsense-mediated mRNA decay, the accelerated turnover of mRNAs transcribed from genes containing early nonsense mutations, is dependent on the product of the UPF1 gene in yeast. Mutations that inactivate UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations but have no effect on the half-lives of almost all other mRNAs. Since the transcripts of nonsense alleles are not typical cellular constituents, we sought to identify those RNAs that comprise normal substrates of the nonsense-mediated mRNA decay pathway. Many yeast pre-mRNAs contain early in-frame nonsense codons and we consider it possible that a role of this pathway is to accelerate the degradation of pre-mRNAs present in the cytoplasm. Consistent with this hypothesis, we find that, in a strain lacking UPF1 function, the CYH2, RP51B, and MER2 pre-mRNAs are stabilized 2- to 5-fold and are associated with ribosomes. We conclude that a major source of early nonsense codon-containing cytoplasmic transcripts in yeast is pre-mRNAs and that the UPF1 protein may be part of a cellular system that ensures that potentially deleterious nonsense fragments of polypeptides do not accumulate.