Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast.
Rapid 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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Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655-0122, USA.
Brown
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Jacobson
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D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020365:RNA Helicases; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; C092470:NMD2 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases