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. 1997 Mar;17(3):1580–1594. doi: 10.1128/mcb.17.3.1580

Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway.

F He 1, A H Brown 1, A Jacobson 1
PMCID: PMC231884  PMID: 9032286

Abstract

Rapid 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.

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Selected References

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  1. Altamura N., Groudinsky O., Dujardin G., Slonimski P. P. NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. J Mol Biol. 1992 Apr 5;224(3):575–587. doi: 10.1016/0022-2836(92)90545-u. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Atkin A. L., Altamura N., Leeds P., Culbertson M. R. The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm. Mol Biol Cell. 1995 May;6(5):611–625. doi: 10.1091/mbc.6.5.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cui Y., Hagan K. W., Zhang S., Peltz S. W. Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon. Genes Dev. 1995 Feb 15;9(4):423–436. doi: 10.1101/gad.9.4.423. [DOI] [PubMed] [Google Scholar]
  5. Culbertson M. R., Underbrink K. M., Fink G. R. Frameshift suppression Saccharomyces cerevisiae. II. Genetic properties of group II suppressors. Genetics. 1980 Aug;95(4):833–853. doi: 10.1093/genetics/95.4.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Czaplinski K., Weng Y., Hagan K. W., Peltz S. W. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. RNA. 1995 Aug;1(6):610–623. [PMC free article] [PubMed] [Google Scholar]
  7. Dinman J. D., Wickner R. B. Translational maintenance of frame: mutants of Saccharomyces cerevisiae with altered -1 ribosomal frameshifting efficiencies. Genetics. 1994 Jan;136(1):75–86. doi: 10.1093/genetics/136.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  9. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  10. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  11. He F., Brown A. H., Jacobson A. Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast. RNA. 1996 Feb;2(2):153–170. [PMC free article] [PubMed] [Google Scholar]
  12. He F., Jacobson A. Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen. Genes Dev. 1995 Feb 15;9(4):437–454. doi: 10.1101/gad.9.4.437. [DOI] [PubMed] [Google Scholar]
  13. He F., Peltz S. W., Donahue J. L., Rosbash M., Jacobson A. Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7034–7038. doi: 10.1073/pnas.90.15.7034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hollenberg S. M., Sternglanz R., Cheng P. F., Weintraub H. Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol Cell Biol. 1995 Jul;15(7):3813–3822. doi: 10.1128/mcb.15.7.3813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
  16. Karlin S. Statistical significance of sequence patterns in proteins. Curr Opin Struct Biol. 1995 Jun;5(3):360–371. doi: 10.1016/0959-440x(95)80098-0. [DOI] [PubMed] [Google Scholar]
  17. Koonin E. V. A new group of putative RNA helicases. Trends Biochem Sci. 1992 Dec;17(12):495–497. doi: 10.1016/0968-0004(92)90338-a. [DOI] [PubMed] [Google Scholar]
  18. Lee B. S., Culbertson M. R. Identification of an additional gene required for eukaryotic nonsense mRNA turnover. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10354–10358. doi: 10.1073/pnas.92.22.10354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee S. I., Umen J. G., Varmus H. E. A genetic screen identifies cellular factors involved in retroviral -1 frameshifting. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6587–6591. doi: 10.1073/pnas.92.14.6587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leeds P., Peltz S. W., Jacobson A., Culbertson M. R. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 1991 Dec;5(12A):2303–2314. doi: 10.1101/gad.5.12a.2303. [DOI] [PubMed] [Google Scholar]
  21. Leeds P., Wood J. M., Lee B. S., Culbertson M. R. Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2165–2177. doi: 10.1128/mcb.12.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maddock J. R., Weidenhammer E. M., Adams C. C., Lunz R. L., Woolford J. L., Jr Extragenic suppressors of Saccharomyces cerevisiae prp4 mutations identify a negative regulator of PRP genes. Genetics. 1994 Mar;136(3):833–847. doi: 10.1093/genetics/136.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Peltz S. W., Brown A. H., Jacobson A. mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor. Genes Dev. 1993 Sep;7(9):1737–1754. doi: 10.1101/gad.7.9.1737. [DOI] [PubMed] [Google Scholar]
  24. Peltz S. W., He F., Welch E., Jacobson A. Nonsense-mediated mRNA decay in yeast. Prog Nucleic Acid Res Mol Biol. 1994;47:271–298. doi: 10.1016/s0079-6603(08)60254-8. [DOI] [PubMed] [Google Scholar]
  25. Pinto I., Na J. G., Sherman F., Hampsey M. cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae. Genetics. 1992 Sep;132(1):97–112. doi: 10.1093/genetics/132.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rodgers K. K., Bu Z., Fleming K. G., Schatz D. G., Engelman D. M., Coleman J. E. A zinc-binding domain involved in the dimerization of RAG1. J Mol Biol. 1996 Jul 5;260(1):70–84. doi: 10.1006/jmbi.1996.0382. [DOI] [PubMed] [Google Scholar]
  27. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  30. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thompson E. A., Roeder G. S. Expression and DNA sequence of RED1, a gene required for meiosis I chromosome segregation in yeast. Mol Gen Genet. 1989 Aug;218(2):293–301. doi: 10.1007/BF00331281. [DOI] [PubMed] [Google Scholar]
  32. Weng Y., Czaplinski K., Peltz S. W. Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol Cell Biol. 1996 Oct;16(10):5491–5506. doi: 10.1128/mcb.16.10.5491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. White T. J., Arnheim N., Erlich H. A. The polymerase chain reaction. Trends Genet. 1989 Jun;5(6):185–189. doi: 10.1016/0168-9525(89)90073-5. [DOI] [PubMed] [Google Scholar]
  34. Zervos A. S., Gyuris J., Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell. 1993 Jan 29;72(2):223–232. doi: 10.1016/0092-8674(93)90662-a. [DOI] [PubMed] [Google Scholar]
  35. Zhang S., Ruiz-Echevarria M. J., Quan Y., Peltz S. W. Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay. Mol Cell Biol. 1995 Apr;15(4):2231–2244. doi: 10.1128/mcb.15.4.2231. [DOI] [PMC free article] [PubMed] [Google Scholar]

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