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. 1997 Jun;17(6):3398–3407. doi: 10.1128/mcb.17.6.3398

Rok1p is a putative RNA helicase required for rRNA processing.

J Venema 1, C Bousquet-Antonelli 1, J P Gelugne 1, M Caizergues-Ferrer 1, D Tollervey 1
PMCID: PMC232193  PMID: 9154839

Abstract

The synthesis of ribosomes involves many small nucleolar ribonucleoprotein particles (snoRNPs) as transacting factors. Yeast strains lacking the snoRNA, snR10, are viable but are impaired in growth and delayed in the early pre-rRNA cleavages at sites A0, A1, and A2, which lead to the synthesis of 18S rRNA. The same cleavages are inhibited by genetic depletion of the essential snoRNP protein Gar1p. Screens for mutations showing synthetic lethality with deletion of the SNR10 gene or with a temperature-sensitive gar1 allele both identified the ROK1 gene, encoding a putative, ATP-dependent RNA helicase of the DEAD-box family. The ROK1 gene is essential for viability, and depletion of Rok1p inhibits pre-rRNA processing at sites A0, A1, and A2, thereby blocking 18S rRNA synthesis. Indirect immunofluorescence by using a ProtA-Rok1p construct shows the protein to be predominantly nucleolar. These results suggest that Rok1p is required for the function of the snoRNP complex carrying out the early pre-rRNA cleavage reactions.

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Selected References

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  1. Amberg D. C., Goldstein A. L., Cole C. N. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 1992 Jul;6(7):1173–1189. doi: 10.1101/gad.6.7.1173. [DOI] [PubMed] [Google Scholar]
  2. Aris J. P., Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J Cell Biol. 1988 Jul;107(1):17–31. doi: 10.1083/jcb.107.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balakin A. G., Smith L., Fournier M. J. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell. 1996 Sep 6;86(5):823–834. doi: 10.1016/s0092-8674(00)80156-7. [DOI] [PubMed] [Google Scholar]
  4. Beltrame M., Henry Y., Tollervey D. Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA. Nucleic Acids Res. 1994 Nov 25;22(23):5139–5147. doi: 10.1093/nar/22.23.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beltrame M., Tollervey D. Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J. 1995 Sep 1;14(17):4350–4356. doi: 10.1002/j.1460-2075.1995.tb00109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beltrame M., Tollervey D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J. 1992 Apr;11(4):1531–1542. doi: 10.1002/j.1460-2075.1992.tb05198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berben G., Dumont J., Gilliquet V., Bolle P. A., Hilger F. The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast. 1991 Jul;7(5):475–477. doi: 10.1002/yea.320070506. [DOI] [PubMed] [Google Scholar]
  8. Bergès T., Petfalski E., Tollervey D., Hurt E. C. Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification. EMBO J. 1994 Jul 1;13(13):3136–3148. doi: 10.1002/j.1460-2075.1994.tb06612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  10. Chu S., Archer R. H., Zengel J. M., Lindahl L. The RNA of RNase MRP is required for normal processing of ribosomal RNA. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):659–663. doi: 10.1073/pnas.91.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dammel C. S., Noller H. F. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 1993 Apr;7(4):660–670. doi: 10.1101/gad.7.4.660. [DOI] [PubMed] [Google Scholar]
  12. Eichler D. C., Craig N. Processing of eukaryotic ribosomal RNA. Prog Nucleic Acid Res Mol Biol. 1994;49:197–239. doi: 10.1016/s0079-6603(08)60051-3. [DOI] [PubMed] [Google Scholar]
  13. Elela S. A., Igel H., Ares M., Jr RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. Cell. 1996 Apr 5;85(1):115–124. doi: 10.1016/s0092-8674(00)81087-9. [DOI] [PubMed] [Google Scholar]
  14. Feunteun J., Monier R., Vola C., Rosset R. Ribosomal assembly defective mutants of Escherichia coli. Nucleic Acids Res. 1974 Jan;1(1):149–169. doi: 10.1093/nar/1.1.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuller-Pace F. V., Nicol S. M., Reid A. D., Lane D. P. DbpA: a DEAD box protein specifically activated by 23s rRNA. EMBO J. 1993 Sep;12(9):3619–3626. doi: 10.1002/j.1460-2075.1993.tb06035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fuller-Pace F. V. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. doi: 10.1016/0962-8924(94)90210-0. [DOI] [PubMed] [Google Scholar]
  17. Ghisolfi L., Joseph G., Amalric F., Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed] [Google Scholar]
  18. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Girard J. P., Bagni C., Caizergues-Ferrer M., Amalric F., Lapeyre B. Identification of a segment of the small nucleolar ribonucleoprotein-associated protein GAR1 that is sufficient for nucleolar accumulation. J Biol Chem. 1994 Jul 15;269(28):18499–18506. [PubMed] [Google Scholar]
  21. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grandi P., Doye V., Hurt E. C. Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96. EMBO J. 1993 Aug;12(8):3061–3071. doi: 10.1002/j.1460-2075.1993.tb05975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gururajan R., Mathews L., Longo F. J., Weeks D. L. An3 mRNA encodes an RNA helicase that colocalizes with nucleoli in Xenopus oocytes in a stage-specific manner. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2056–2060. doi: 10.1073/pnas.91.6.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guthrie C., Nashimoto H., Nomura M. Structure and function of E. coli ribosomes. 8. Cold-sensitive mutants defective in ribosome assembly. Proc Natl Acad Sci U S A. 1969 Jun;63(2):384–391. doi: 10.1073/pnas.63.2.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henry Y., Wood H., Morrissey J. P., Petfalski E., Kearsey S., Tollervey D. The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site. EMBO J. 1994 May 15;13(10):2452–2463. doi: 10.1002/j.1460-2075.1994.tb06530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Hughes J. M., Ares M., Jr Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 1991 Dec;10(13):4231–4239. doi: 10.1002/j.1460-2075.1991.tb05001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Interthal H., Bellocq C., Bähler J., Bashkirov V. I., Edelstein S., Heyer W. D. A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae. EMBO J. 1995 Mar 15;14(6):1057–1066. doi: 10.1002/j.1460-2075.1995.tb07088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jansen R., Tollervey D., Hurt E. C. A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing. EMBO J. 1993 Jun;12(6):2549–2558. doi: 10.1002/j.1460-2075.1993.tb05910.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kearsey S., Kipling D. Recombination and RNA processing: a common strand? Trends Cell Biol. 1991 Nov;1(5):110–112. doi: 10.1016/0962-8924(91)90101-e. [DOI] [PubMed] [Google Scholar]
  32. Koshland D., Kent J. C., Hartwell L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. doi: 10.1016/0092-8674(85)90153-9. [DOI] [PubMed] [Google Scholar]
  33. Kranz J. E., Holm C. Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6629–6633. doi: 10.1073/pnas.87.17.6629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lafontaine D., Tollervey D. Trans-acting factors in yeast pre-rRNA and pre-snoRNA processing. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):803–812. doi: 10.1139/o95-088. [DOI] [PubMed] [Google Scholar]
  35. Laggerbauer B., Lauber J., Lührmann R. Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs. Nucleic Acids Res. 1996 Mar 1;24(5):868–875. doi: 10.1093/nar/24.5.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Larimer F. W., Hsu C. L., Maupin M. K., Stevens A. Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells. Gene. 1992 Oct 12;120(1):51–57. doi: 10.1016/0378-1119(92)90008-d. [DOI] [PubMed] [Google Scholar]
  37. Larimer F. W., Stevens A. Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth. Gene. 1990 Oct 30;95(1):85–90. doi: 10.1016/0378-1119(90)90417-p. [DOI] [PubMed] [Google Scholar]
  38. Li H. D., Zagorski J., Fournier M. J. Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Mar;10(3):1145–1152. doi: 10.1128/mcb.10.3.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Liang W. Q., Fournier M. J. U14 base-pairs with 18S rRNA: a novel snoRNA interaction required for rRNA processing. Genes Dev. 1995 Oct 1;9(19):2433–2443. doi: 10.1101/gad.9.19.2433. [DOI] [PubMed] [Google Scholar]
  40. Lygerou Z., Allmang C., Tollervey D., Séraphin B. Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro. Science. 1996 Apr 12;272(5259):268–270. doi: 10.1126/science.272.5259.268. [DOI] [PubMed] [Google Scholar]
  41. Lygerou Z., Mitchell P., Petfalski E., Séraphin B., Tollervey D. The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins. Genes Dev. 1994 Jun 15;8(12):1423–1433. doi: 10.1101/gad.8.12.1423. [DOI] [PubMed] [Google Scholar]
  42. Maxwell E. S., Fournier M. J. The small nucleolar RNAs. Annu Rev Biochem. 1995;64:897–934. doi: 10.1146/annurev.bi.64.070195.004341. [DOI] [PubMed] [Google Scholar]
  43. Morrissey J. P., Tollervey D. Birth of the snoRNPs: the evolution of RNase MRP and the eukaryotic pre-rRNA-processing system. Trends Biochem Sci. 1995 Feb;20(2):78–82. doi: 10.1016/s0968-0004(00)88962-8. [DOI] [PubMed] [Google Scholar]
  44. Morrissey J. P., Tollervey D. Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis. Mol Cell Biol. 1993 Apr;13(4):2469–2477. doi: 10.1128/mcb.13.4.2469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Mélèse T., Xue Z. The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol. 1995 Jun;7(3):319–324. doi: 10.1016/0955-0674(95)80085-9. [DOI] [PubMed] [Google Scholar]
  47. Nicol S. M., Fuller-Pace F. V. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11681–11685. doi: 10.1073/pnas.92.25.11681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nishi K., Morel-Deville F., Hershey J. W., Leighton T., Schnier J. An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly. Nature. 1988 Dec 1;336(6198):496–498. doi: 10.1038/336496a0. [DOI] [PubMed] [Google Scholar]
  49. O'Day C. L., Chavanikamannil F., Abelson J. 18S rRNA processing requires the RNA helicase-like protein Rrp3. Nucleic Acids Res. 1996 Aug 15;24(16):3201–3207. doi: 10.1093/nar/24.16.3201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ripmaster T. L., Vaughn G. P., Woolford J. L., Jr A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11131–11135. doi: 10.1073/pnas.89.23.11131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Sachs A. B., Davis R. W. Translation initiation and ribosomal biogenesis: involvement of a putative rRNA helicase and RPL46. Science. 1990 Mar 2;247(4946):1077–1079. doi: 10.1126/science.2408148. [DOI] [PubMed] [Google Scholar]
  53. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  54. Schmitt M. E., Clayton D. A. Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7935–7941. doi: 10.1128/mcb.13.12.7935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Song Y., Kim S., Kim J. ROK1, a high-copy-number plasmid suppressor of kem1, encodes a putative ATP-dependent RNA helicase in Saccharomyces cerevisiae. Gene. 1995 Dec 1;166(1):151–154. doi: 10.1016/0378-1119(96)80010-2. [DOI] [PubMed] [Google Scholar]
  56. Stevens A., Hsu C. L., Isham K. R., Larimer F. W. Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1. J Bacteriol. 1991 Nov;173(21):7024–7028. doi: 10.1128/jb.173.21.7024-7028.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Stevens A., Poole T. L. 5'-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5'-exonuclease-1. J Biol Chem. 1995 Jul 7;270(27):16063–16069. doi: 10.1074/jbc.270.27.16063. [DOI] [PubMed] [Google Scholar]
  58. Tollervey D. A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 1987 Dec 20;6(13):4169–4175. doi: 10.1002/j.1460-2075.1987.tb02763.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tollervey D., Guthrie C. Deletion of a yeast small nuclear RNA gene impairs growth. EMBO J. 1985 Dec 30;4(13B):3873–3878. doi: 10.1002/j.1460-2075.1985.tb04160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tollervey D., Lehtonen H., Carmo-Fonseca M., Hurt E. C. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 1991 Mar;10(3):573–583. doi: 10.1002/j.1460-2075.1991.tb07984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Toone W. M., Rudd K. E., Friesen J. D. deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2. J Bacteriol. 1991 Jun;173(11):3291–3302. doi: 10.1128/jb.173.11.3291-3302.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tuteja N., Huang N. W., Skopac D., Tuteja R., Hrvatic S., Zhang J., Pongor S., Joseph G., Faucher C., Amalric F. Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation. Gene. 1995 Jul 28;160(2):143–148. doi: 10.1016/0378-1119(95)00207-m. [DOI] [PubMed] [Google Scholar]
  63. Venema J., Tollervey D. Processing of pre-ribosomal RNA in Saccharomyces cerevisiae. Yeast. 1995 Dec;11(16):1629–1650. doi: 10.1002/yea.320111607. [DOI] [PubMed] [Google Scholar]
  64. 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]

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