Browsing by keyword "*RNA Helicases"
Now showing items 1-4 of 4
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Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNAThe determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.
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Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screenRapid 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.
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Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeastRapid turnover of nonsense-containing mRNAs in the yeast Saccharomyces cerevisiae is dependent on the products of the UPF1 (Upf1p), NMD2/UPF2 (Nmd2p) and UPF3 (Upf3p) genes. Mutations in each of these genes lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. NMD2 was recently identified in a two-hybrid screen as a gene that encodes a Upf1p-interacting protein. To identify the amino acids essential to this interaction, we used two-hybrid analysis as well as missense, nonsense, and deletion mutants of NMD2, and mapped the Upf1p-interacting domain of Nmd2p to a 157-amino acid segment at its C-terminus. Mutations in this domain that disrupt interaction with Upf1p also disrupt nonsense-mediated mRNA decay. A dominant-negative deletion allele of NMD2 identified previously includes the Upf1p-interacting domain. However, mutations in the Upf1p-interacting domain do not affect dominant-negative inhibition of mRNA decay caused by this allele, suggesting interaction with yet another factor. These results, and the observation that deletion of a putative nuclear localization signal and a putative transmembrane domain also inactivate nonsense-mediated mRNA decay, suggest that Nmd2p may contain as many as four important functional domains.
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Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathwayRapid turnover of nonsense-containing mRNAs in Saccharomyces cerevisiae is dependent on Upf1p, Nmd2p, and Upf3p, the products of the UPF1, NMD2/UPF2, and UPF3 genes, respectively. We showed previously that Upf1p and Nmd2p interact and that this interaction is required for nonsense-mediated mRNA decay (F. He and A. Jacobson, Genes Dev. 9:437-454, 1995; F. He, A. H. Brown, and A. Jacobson, RNA 2:153-170, 1996). In this study we have used the yeast two-hybrid system to define other protein-protein interactions among the essential components of this decay pathway. Nmd2p-Upf3p and Upf1p-Upf3p interactions were identified, and the respective domains involved in these interactions were delineated by deletion analysis. The domains of Upf1p and Upf3p putatively involved in their mutual interaction were found to correspond to the domains on the two proteins which interact with Nmd2p, suggesting that Nmd2p bridges Upf1p and Upf3p. This conclusion was reinforced by experiments showing that: (i) deletion of NMD2 completely abolishes interactions between Upf1p and Upf3p and (ii) overexpression of full-length Nmd2p or Nmd2p fragments that retain Upf1p- and Upf3p-interacting domains promotes 10- to 200-fold enhancement of Upf1p-Nmd2p-Upf3p complex formation. These results; the observation that cells harboring either single or multiple deletions of UPF1, NMD2, and UPF3 inhibit nonsense-mediated mRNA decay to the same extent; and an analysis of the possible targets of a dominant-negative NMD2 allele indicate that Upf1p, Nmd2p, Upf3p, and at least one other factor are functionally dependent, interacting components of the yeast nonsense-mediated mRNA decay pathway.
