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. 1999 Aug;5(8):1055–1070. doi: 10.1017/s1355838299990027

Overexpression of truncated Nmd3p inhibits protein synthesis in yeast.

J P Belk 1, F He 1, A Jacobson 1
PMCID: PMC1369829  PMID: 10445880

Abstract

The yeast NMD3 gene was identified in a two-hybrid screen using the nonsense-mediated mRNA decay factor, Upf1p, as bait. NMD3 was shown to encode an essential, highly conserved protein that associated principally with free 60S ribosomal subunits. Overexpression of a truncated form of Nmd3p, lacking 100 C-terminal amino acids and most of its Upf1p-interacting domain, had dominant-negative effects on both cell growth and protein synthesis and promoted the formation of polyribosome half-mers. These effects were eliminated by truncation of an additional 100 amino acids from Nmd3p. Overexpression of the nmd3delta100 allele also led to increased synthesis and destabilization of some ribosomal protein mRNAs, and increased synthesis and altered processing of 35S pre-rRNA. Our data suggest that Nmd3p has a role in the formation, function, or maintenance of the 60S ribosomal subunit and may provide a link for Upf1p to 80S monosomes.

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Selected References

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  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Atkin A. L., Schenkman L. R., Eastham M., Dahlseid J. N., Lelivelt M. J., Culbertson M. R. Relationship between yeast polyribosomes and Upf proteins required for nonsense mRNA decay. J Biol Chem. 1997 Aug 29;272(35):22163–22172. doi: 10.1074/jbc.272.35.22163. [DOI] [PubMed] [Google Scholar]
  4. Beelman C. A., Stevens A., Caponigro G., LaGrandeur T. E., Hatfield L., Fortner D. M., Parker R. An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature. 1996 Aug 15;382(6592):642–646. doi: 10.1038/382642a0. [DOI] [PubMed] [Google Scholar]
  5. Boorstein W. R., Craig E. A. Primer extension analysis of RNA. Methods Enzymol. 1989;180:347–369. doi: 10.1016/0076-6879(89)80111-9. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Czaplinski K., Ruiz-Echevarria M. J., Paushkin S. V., Han X., Weng Y., Perlick H. A., Dietz H. C., Ter-Avanesyan M. D., Peltz S. W. The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs. Genes Dev. 1998 Jun 1;12(11):1665–1677. doi: 10.1101/gad.12.11.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Decker C. J., Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 1993 Aug;7(8):1632–1643. doi: 10.1101/gad.7.8.1632. [DOI] [PubMed] [Google Scholar]
  10. Decker C. J., Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci. 1994 Aug;19(8):336–340. doi: 10.1016/0968-0004(94)90073-6. [DOI] [PubMed] [Google Scholar]
  11. Dick F. A., Karamanou S., Trumpower B. L. QSR1, an essential yeast gene with a genetic relationship to a subunit of the mitochondrial cytochrome bc1 complex, codes for a 60 S ribosomal subunit protein. J Biol Chem. 1997 May 16;272(20):13372–13379. doi: 10.1074/jbc.272.20.13372. [DOI] [PubMed] [Google Scholar]
  12. Dick F. A., Trumpower B. L. Heterologous complementation reveals that mutant alleles of QSR1 render 60S ribosomal subunits unstable and translationally inactive. Nucleic Acids Res. 1998 May 15;26(10):2442–2448. doi: 10.1093/nar/26.10.2442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eisinger D. P., Dick F. A., Trumpower B. L. Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits. Mol Cell Biol. 1997 Sep;17(9):5136–5145. doi: 10.1128/mcb.17.9.5136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Fried H. M., Nam H. G., Loechel S., Teem J. Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcm1 and cyh2. Mol Cell Biol. 1985 Jan;5(1):99–108. doi: 10.1128/mcb.5.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. He F., Brown A. H., Jacobson A. Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway. Mol Cell Biol. 1997 Mar;17(3):1580–1594. doi: 10.1128/mcb.17.3.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Helser T. L., Baan R. A., Dahlberg A. E. Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide. Mol Cell Biol. 1981 Jan;1(1):51–57. doi: 10.1128/mcb.1.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herrick D., Parker R., Jacobson A. Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2269–2284. doi: 10.1128/mcb.10.5.2269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ho J. H., Johnson A. W. NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Mar;19(3):2389–2399. doi: 10.1128/mcb.19.3.2389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hodges P. E., McKee A. H., Davis B. P., Payne W. E., Garrels J. I. The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data. Nucleic Acids Res. 1999 Jan 1;27(1):69–73. doi: 10.1093/nar/27.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hsu C. L., Stevens A. Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure. Mol Cell Biol. 1993 Aug;13(8):4826–4835. doi: 10.1128/mcb.13.8.4826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. LaGrandeur T. E., Parker R. Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme. EMBO J. 1998 Mar 2;17(5):1487–1496. doi: 10.1093/emboj/17.5.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Mager W. H., Planta R. J., Ballesta J. G., Lee J. C., Mizuta K., Suzuki K., Warner J. R., Woolford J. A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Nucleic Acids Res. 1997 Dec 15;25(24):4872–4875. doi: 10.1093/nar/25.24.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mangus D. A., Jacobson A. Linking mRNA turnover and translation: assessing the polyribosomal association of mRNA decay factors and degradative intermediates. Methods. 1999 Jan;17(1):28–37. doi: 10.1006/meth.1998.0704. [DOI] [PubMed] [Google Scholar]
  33. Mizuta K., Tsujii R., Warner J. R., Nishiyama M. The C-terminal silencing domain of Rap1p is essential for the repression of ribosomal protein genes in response to a defect in the secretory pathway. Nucleic Acids Res. 1998 Feb 15;26(4):1063–1069. doi: 10.1093/nar/26.4.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moehle C. M., Hinnebusch A. G. Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 May;11(5):2723–2735. doi: 10.1128/mcb.11.5.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moritz M., Pulaski B. A., Woolford J. L., Jr Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16. Mol Cell Biol. 1991 Nov;11(11):5681–5692. doi: 10.1128/mcb.11.11.5681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Muhlrad D., Decker C. J., Parker R. Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes Dev. 1994 Apr 1;8(7):855–866. doi: 10.1101/gad.8.7.855. [DOI] [PubMed] [Google Scholar]
  37. Muhlrad D., Decker C. J., Parker R. Turnover mechanisms of the stable yeast PGK1 mRNA. Mol Cell Biol. 1995 Apr;15(4):2145–2156. doi: 10.1128/mcb.15.4.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Muhlrad D., Parker R. Premature translational termination triggers mRNA decapping. Nature. 1994 Aug 18;370(6490):578–581. doi: 10.1038/370578a0. [DOI] [PubMed] [Google Scholar]
  39. Nam H. G., Fried H. M. Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation. Mol Cell Biol. 1986 May;6(5):1535–1544. doi: 10.1128/mcb.6.5.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nelson E. M., Winkler M. M. Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes. J Biol Chem. 1987 Aug 25;262(24):11501–11506. [PubMed] [Google Scholar]
  41. Ohtake Y., Wickner R. B. Yeast virus propagation depends critically on free 60S ribosomal subunit concentration. Mol Cell Biol. 1995 May;15(5):2772–2781. doi: 10.1128/mcb.15.5.2772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  44. Rotenberg M. O., Moritz M., Woolford J. L., Jr Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes. Genes Dev. 1988 Feb;2(2):160–172. doi: 10.1101/gad.2.2.160. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Stevens A. mRNA-decapping enzyme from Saccharomyces cerevisiae: purification and unique specificity for long RNA chains. Mol Cell Biol. 1988 May;8(5):2005–2010. doi: 10.1128/mcb.8.5.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sun X., Perlick H. A., Dietz H. C., Maquat L. E. A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10009–10014. doi: 10.1073/pnas.95.17.10009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  51. Ulery T. L., Mangus D. A., Jaehning J. A. The yeast IMP1 gene is allelic to GAL2. Mol Gen Genet. 1991 Nov;230(1-2):129–135. doi: 10.1007/BF00290660. [DOI] [PubMed] [Google Scholar]
  52. Venema J., Tollervey D. RRP5 is required for formation of both 18S and 5.8S rRNA in yeast. EMBO J. 1996 Oct 15;15(20):5701–5714. [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Zuk D., Jacobson A. A single amino acid substitution in yeast eIF-5A results in mRNA stabilization. EMBO J. 1998 May 15;17(10):2914–2925. doi: 10.1093/emboj/17.10.2914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. van Venrooij W. J., van Eenbergen J., Janssen A. P. Effect of anisomycin on the cellular level of native ribosomal subunits. Biochemistry. 1977 May 31;16(11):2343–2348. doi: 10.1021/bi00630a006. [DOI] [PubMed] [Google Scholar]

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