Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Jul 3;92(14):6587–6591. doi: 10.1073/pnas.92.14.6587

A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.

S I Lee 1, J G Umen 1, H E Varmus 1
PMCID: PMC41563  PMID: 7604038

Abstract

To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.

Full text

PDF
6587

Images in this article

6588Fig. 1
on p.6588
6589Fig. 2
on p.6589
6590Fig. 3
on p.6590
6590Fig. 5
on p.6590

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alksne L. E., Anthony R. A., Liebman S. W., Warner J. R. An accuracy center in the ribosome conserved over 2 billion years. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9538–9541. doi: 10.1073/pnas.90.20.9538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. All-Robyn J. A., Brown N., Otaka E., Liebman S. W. Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein. Mol Cell Biol. 1990 Dec;10(12):6544–6553. doi: 10.1128/mcb.10.12.6544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atkins J. F., Weiss R. B., Gesteland R. F. Ribosome gymnastics--degree of difficulty 9.5, style 10.0. Cell. 1990 Aug 10;62(3):413–423. doi: 10.1016/0092-8674(90)90007-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bollen G. H., Mager W. H., Planta R. J. High resolution mini-two-dimensional gel electrophoresis of yeast ribosomal proteins. A standard nomenclature for yeast ribosomal proteins. Mol Biol Rep. 1981 Nov 30;8(1):37–44. doi: 10.1007/BF00798383. [DOI] [PubMed] [Google Scholar]
  5. Breeden L., Nasmyth K. Cell cycle control of the yeast HO gene: cis- and trans-acting regulators. Cell. 1987 Feb 13;48(3):389–397. doi: 10.1016/0092-8674(87)90190-5. [DOI] [PubMed] [Google Scholar]
  6. Brierley I., Digard P., Inglis S. C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell. 1989 May 19;57(4):537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caizergues-Ferrer M., Mariottini P., Curie C., Lapeyre B., Gas N., Amalric F., Amaldi F. Nucleolin from Xenopus laevis: cDNA cloning and expression during development. Genes Dev. 1989 Mar;3(3):324–333. doi: 10.1101/gad.3.3.324. [DOI] [PubMed] [Google Scholar]
  8. Chamorro M., Parkin N., Varmus H. E. An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):713–717. doi: 10.1073/pnas.89.2.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen X., Chamorro M., Lee S. I., Shen L. X., Hines J. V., Tinoco I., Jr, Varmus H. E. Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting. EMBO J. 1995 Feb 15;14(4):842–852. doi: 10.1002/j.1460-2075.1995.tb07062.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Culbertson M. R., Gaber R. F., Cummins C. M. Frameshift suppression in Saccharomyces cerevisiae. V. Isolation and genetic properties of nongroup-specific suppressors. Genetics. 1982 Nov;102(3):361–378. doi: 10.1093/genetics/102.3.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Felsenstein K. M., Goff S. P. Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing. J Virol. 1988 Jun;62(6):2179–2182. doi: 10.1128/jvi.62.6.2179-2182.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fogel S., Welch J. W. Tandem gene amplification mediates copper resistance in yeast. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5342–5346. doi: 10.1073/pnas.79.17.5342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gulyas K. D., Donahue T. F. SSL2, a suppressor of a stem-loop mutation in the HIS4 leader encodes the yeast homolog of human ERCC-3. Cell. 1992 Jun 12;69(6):1031–1042. doi: 10.1016/0092-8674(92)90621-i. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988 Nov 4;55(3):447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  20. Jacks T. Translational suppression in gene expression in retroviruses and retrotransposons. Curr Top Microbiol Immunol. 1990;157:93–124. doi: 10.1007/978-3-642-75218-6_4. [DOI] [PubMed] [Google Scholar]
  21. Larkin J. C., Thompson J. R., Woolford J. L., Jr Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene. Mol Cell Biol. 1987 May;7(5):1764–1775. doi: 10.1128/mcb.7.5.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lesser C. F., Guthrie C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics. 1993 Apr;133(4):851–863. doi: 10.1093/genetics/133.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Parkin N. T., Chamorro M., Varmus H. E. Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo. J Virol. 1992 Aug;66(8):5147–5151. doi: 10.1128/jvi.66.8.5147-5151.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Puglisi J. D., Wyatt J. R., Tinoco I., Jr Conformation of an RNA pseudoknot. J Mol Biol. 1990 Jul 20;214(2):437–453. doi: 10.1016/0022-2836(90)90192-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Sandbaken M. G., Culbertson M. R. Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae. Genetics. 1988 Dec;120(4):923–934. doi: 10.1093/genetics/120.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schmid S. R., Linder P. Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases. Mol Cell Biol. 1991 Jul;11(7):3463–3471. doi: 10.1128/mcb.11.7.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schmidt-Zachmann M. S., Nigg E. A. Protein localization to the nucleolus: a search for targeting domains in nucleolin. J Cell Sci. 1993 Jul;105(Pt 3):799–806. doi: 10.1242/jcs.105.3.799. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Vincent A., Liebman S. W. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein. Genetics. 1992 Oct;132(2):375–386. doi: 10.1093/genetics/132.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson P. G., Culbertson M. R. SUF12 suppressor protein of yeast. A fusion protein related to the EF-1 family of elongation factors. J Mol Biol. 1988 Feb 20;199(4):559–573. doi: 10.1016/0022-2836(88)90301-4. [DOI] [PubMed] [Google Scholar]
  32. Wilson W., Braddock M., Adams S. E., Rathjen P. D., Kingsman S. M., Kingsman A. J. HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell. 1988 Dec 23;55(6):1159–1169. doi: 10.1016/0092-8674(88)90260-7. [DOI] [PubMed] [Google Scholar]
  33. ten Dam E. B., Pleij C. W., Bosch L. RNA pseudoknots: translational frameshifting and readthrough on viral RNAs. Virus Genes. 1990 Jul;4(2):121–136. doi: 10.1007/BF00678404. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES