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. 1995 May;15(5):2772–2781. doi: 10.1128/mcb.15.5.2772

Yeast virus propagation depends critically on free 60S ribosomal subunit concentration.

Y Ohtake 1, R B Wickner 1
PMCID: PMC230508  PMID: 7739558

Abstract

Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3' poly(A).

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Selected References

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  1. Abovich N., Gritz L., Tung L., Rosbash M. Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Dec;5(12):3429–3435. doi: 10.1128/mcb.5.12.3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abovich N., Rosbash M. Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes. Mol Cell Biol. 1984 Sep;4(9):1871–1879. doi: 10.1128/mcb.4.9.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alksne L. E., Warner J. R. A novel cloning strategy reveals the gene for the yeast homologue to Escherichia coli ribosomal protein S12. J Biol Chem. 1993 May 25;268(15):10813–10819. [PubMed] [Google Scholar]
  4. Arevalo S. G., Warner J. R. Ribosomal protein L4 of Saccharomyces cerevisiae: the gene and its protein. Nucleic Acids Res. 1990 Mar 25;18(6):1447–1449. doi: 10.1093/nar/18.6.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ball S. G., Tirtiaux C., Wickner R. B. Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE. Genetics. 1984 Jun;107(2):199–217. doi: 10.1093/genetics/107.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanc A., Ribas J. C., Wickner R. B., Sonenberg N. His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression. Mol Cell Biol. 1994 Apr;14(4):2664–2674. doi: 10.1128/mcb.14.4.2664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bostian K. A., Sturgeon J. A., Tipper D. J. Encapsidation of yeast killer double-stranded ribonucleic acids: dependence of M on L. J Bacteriol. 1980 Jul;143(1):463–470. doi: 10.1128/jb.143.1.463-470.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  10. Bruenn J. A., Brennan V. E. Yeast viral double-stranded RNAs have heterogeneous 3' termini. Cell. 1980 Apr;19(4):923–933. doi: 10.1016/0092-8674(80)90084-7. [DOI] [PubMed] [Google Scholar]
  11. Bruenn J., Keitz B. The 5' ends of yeast killer factor RNAs are pppGp. Nucleic Acids Res. 1976 Oct;3(10):2427–2436. doi: 10.1093/nar/3.10.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cigan A. M., Bushman J. L., Boal T. R., Hinnebusch A. G. A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5350–5354. doi: 10.1073/pnas.90.11.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dinman J. D., Icho T., Wickner R. B. A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):174–178. doi: 10.1073/pnas.88.1.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dinman J. D., Wickner R. B. Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation. J Virol. 1992 Jun;66(6):3669–3676. doi: 10.1128/jvi.66.6.3669-3676.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Erickson J. R., Johnston M. Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo. Genetics. 1993 May;134(1):151–157. doi: 10.1093/genetics/134.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Foiani M., Cigan A. M., Paddon C. J., Harashima S., Hinnebusch A. G. GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3203–3216. doi: 10.1128/mcb.11.6.3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hannig E. M., Cigan A. M., Freeman B. A., Kinzy T. G. GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):506–520. doi: 10.1128/mcb.13.1.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harashima S., Hinnebusch A. G. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Nov;6(11):3990–3998. doi: 10.1128/mcb.6.11.3990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartwell L. H., McLaughlin C. S. Temperature-sensitive mutants of yeast exhibiting a rapid inhibition of protein synthesis. J Bacteriol. 1968 Nov;96(5):1664–1671. doi: 10.1128/jb.96.5.1664-1671.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  22. Icho T., Wickner R. B. The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein. J Biol Chem. 1988 Jan 25;263(3):1467–1475. [PubMed] [Google Scholar]
  23. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Jensen R., Sprague G. F., Jr, Herskowitz I. Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus. Proc Natl Acad Sci U S A. 1983 May;80(10):3035–3039. doi: 10.1073/pnas.80.10.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jong A. Y., Clark M. W., Gilbert M., Oehm A., Campbell J. L. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. doi: 10.1128/mcb.7.8.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kang H. A., Hershey J. W. Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J Biol Chem. 1994 Feb 11;269(6):3934–3940. [PubMed] [Google Scholar]
  28. Lee Y. J., Wickner R. B. MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs. Genetics. 1992 Sep;132(1):87–96. doi: 10.1093/genetics/132.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Masison D. C., Blanc A., Ribas J. C., Carroll K., Sonenberg N., Wickner R. B. Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system. Mol Cell Biol. 1995 May;15(5):2763–2771. doi: 10.1128/mcb.15.5.2763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matsumoto Y., Wickner R. B. Yeast 20 S RNA replicon. Replication intermediates and encoded putative RNA polymerase. J Biol Chem. 1991 Jul 5;266(19):12779–12783. [PubMed] [Google Scholar]
  31. Munroe D., Jacobson A. Tales of poly(A): a review. Gene. 1990 Jul 16;91(2):151–158. doi: 10.1016/0378-1119(90)90082-3. [DOI] [PubMed] [Google Scholar]
  32. Ridley S. P., Sommer S. S., Wickner R. B. Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN. Mol Cell Biol. 1984 Apr;4(4):761–770. doi: 10.1128/mcb.4.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  36. Schultz M. C., Brill S. J., Ju Q., Sternglanz R., Reeder R. H. Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation. Genes Dev. 1992 Jul;6(7):1332–1341. doi: 10.1101/gad.6.7.1332. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Sommer S. S., Wickner R. B. Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes [HOK], [NEX] and [EXL] are on one of these dsRNAs. Cell. 1982 Dec;31(2 Pt 1):429–441. doi: 10.1016/0092-8674(82)90136-2. [DOI] [PubMed] [Google Scholar]
  39. Tercero J. C., Dinman J. D., Wickner R. B. Yeast MAK3 N-acetyltransferase recognizes the N-terminal four amino acids of the major coat protein (gag) of the L-A double-stranded RNA virus. J Bacteriol. 1993 May;175(10):3192–3194. doi: 10.1128/jb.175.10.3192-3194.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tercero J. C., Riles L. E., Wickner R. B. Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae. J Biol Chem. 1992 Oct 5;267(28):20270–20276. [PubMed] [Google Scholar]
  41. Tercero J. C., Wickner R. B. MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly. J Biol Chem. 1992 Oct 5;267(28):20277–20281. [PubMed] [Google Scholar]
  42. Thiele D. J., Hannig E. M., Leibowitz M. J. Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation. Mol Cell Biol. 1984 Jan;4(1):92–100. doi: 10.1128/mcb.4.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thrash C., Bankier A. T., Barrell B. G., Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. doi: 10.1073/pnas.82.13.4374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thrash C., Voelkel K., DiNardo S., Sternglanz R. Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem. 1984 Feb 10;259(3):1375–1377. [PubMed] [Google Scholar]
  45. Toh-E A., Wickner R. B. "Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jan;77(1):527–530. doi: 10.1073/pnas.77.1.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tu C., Tzeng T. H., Bruenn J. A. Ribosomal movement impeded at a pseudoknot required for frameshifting. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8636–8640. doi: 10.1073/pnas.89.18.8636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tzeng T. H., Tu C. L., Bruenn J. A. Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus. J Virol. 1992 Feb;66(2):999–1006. doi: 10.1128/jvi.66.2.999-1006.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Uemura H., Wickner R. B. Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein. Mol Cell Biol. 1988 Feb;8(2):938–944. doi: 10.1128/mcb.8.2.938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Valle R. P., Wickner R. B. Elimination of L-A double-stranded RNA virus of Saccharomyces cerevisiae by expression of gag and gag-pol from an L-A cDNA clone. J Virol. 1993 May;67(5):2764–2771. doi: 10.1128/jvi.67.5.2764-2771.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wickner R. B. Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6007–6011. doi: 10.1073/pnas.85.16.6007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wickner R. B., Icho T., Fujimura T., Widner W. R. Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy. J Virol. 1991 Jan;65(1):155–161. doi: 10.1128/jvi.65.1.155-161.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wickner R. B., Koh T. J., Crowley J. C., O'Neil J., Kaback D. B. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures. Yeast. 1987 Mar;3(1):51–57. doi: 10.1002/yea.320030108. [DOI] [PubMed] [Google Scholar]
  53. Wickner R. B., Leibowitz M. J. Chromosomal genes essential for replication of a double-stranded RNA plasmid of Saccharomyces cerevisiae: the killer character of yeast. J Mol Biol. 1976 Aug 15;105(3):427–443. doi: 10.1016/0022-2836(76)90102-9. [DOI] [PubMed] [Google Scholar]
  54. Wickner R. B. Plasmids controlled exclusion of the K2 killer double-stranded RNA plasmid of yeast. Cell. 1980 Aug;21(1):217–226. doi: 10.1016/0092-8674(80)90129-4. [DOI] [PubMed] [Google Scholar]
  55. Wickner R. B., Ridley S. P., Fried H. M., Ball S. G. Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4706–4708. doi: 10.1073/pnas.79.15.4706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Widner W. R., Wickner R. B. Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA. Mol Cell Biol. 1993 Jul;13(7):4331–4341. doi: 10.1128/mcb.13.7.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yon J., Giallongo A., Fried M. The organization and expression of the Saccharomyces cerevisiae L4 ribosomal protein genes and their identification as the homologues of the mammalian ribosomal protein gene L7a. Mol Gen Genet. 1991 May;227(1):72–80. doi: 10.1007/BF00260709. [DOI] [PubMed] [Google Scholar]
  58. Zhong T., Arndt K. T. The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation. Cell. 1993 Jun 18;73(6):1175–1186. doi: 10.1016/0092-8674(93)90646-8. [DOI] [PubMed] [Google Scholar]
  59. Zhu Y. S., Kane J., Zhang X. Y., Zhang M., Tipper D. J. Role of the gamma component of preprotoxin in expression of the yeast K1 killer phenotype. Yeast. 1993 Mar;9(3):251–266. doi: 10.1002/yea.320090305. [DOI] [PubMed] [Google Scholar]

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