Abstract
During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.
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- Allmang C., Henry Y., Morrissey J. P., Wood H., Petfalski E., Tollervey D. Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked. RNA. 1996 Jan;2(1):63–73. [PMC free article] [PubMed] [Google Scholar]
- Allmang C., Henry Y., Wood H., Morrissey J. P., Petfalski E., Tollervey D. Recognition of cleavage site A(2) in the yeast pre-rRNA. RNA. 1996 Jan;2(1):51–62. [PMC free article] [PubMed] [Google Scholar]
- Baudin-Baillieu A., Guillemet E., Cullin C., Lacroute F. Construction of a yeast strain deleted for the TRP1 promoter and coding region that enhances the efficiency of the polymerase chain reaction-disruption method. Yeast. 1997 Mar 30;13(4):353–356. doi: 10.1002/(SICI)1097-0061(19970330)13:4<353::AID-YEA86>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
- Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
- Bonneaud N., Ozier-Kalogeropoulos O., Li G. Y., Labouesse M., Minvielle-Sebastia L., Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. doi: 10.1002/yea.320070609. [DOI] [PubMed] [Google Scholar]
- Chan Y. L., Suzuki K., Olvera J., Wool I. G. Zinc finger-like motifs in rat ribosomal proteins S27 and S29. Nucleic Acids Res. 1993 Feb 11;21(3):649–655. doi: 10.1093/nar/21.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cullin C., Minvielle-Sebastia L. Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins. Yeast. 1994 Jan;10(1):105–112. doi: 10.1002/yea.320100110. [DOI] [PubMed] [Google Scholar]
- Deshmukh M., Tsay Y. F., Paulovich A. G., Woolford J. L., Jr Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits. Mol Cell Biol. 1993 May;13(5):2835–2845. doi: 10.1128/mcb.13.5.2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Finley D., Bartel B., Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Kondo K., Inouye M. Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing. J Biol Chem. 1992 Aug 15;267(23):16252–16258. [PubMed] [Google Scholar]
- Kondo K., Kowalski L. R., Inouye M. Cold shock induction of yeast NSR1 protein and its role in pre-rRNA processing. J Biol Chem. 1992 Aug 15;267(23):16259–16265. [PubMed] [Google Scholar]
- Lafontaine D., Vandenhaute J., Tollervey D. The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev. 1995 Oct 15;9(20):2470–2481. doi: 10.1101/gad.9.20.2470. [DOI] [PubMed] [Google Scholar]
- Lee W. C., Zabetakis D., Mélèse T. NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits. Mol Cell Biol. 1992 Sep;12(9):3865–3871. doi: 10.1128/mcb.12.9.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Lindahl L., Archer R. H., Zengel J. M. Alternate pathways for processing in the internal transcribed spacer 1 in pre-rRNA of Saccharomyces cerevisiae. Nucleic Acids Res. 1994 Dec 11;22(24):5399–5407. doi: 10.1093/nar/22.24.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moritz M., Paulovich A. G., Tsay Y. F., Woolford J. L., Jr Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2261–2274. doi: 10.1083/jcb.111.6.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Oliver S. G., van der Aart Q. J., Agostoni-Carbone M. L., Aigle M., Alberghina L., Alexandraki D., Antoine G., Anwar R., Ballesta J. P., Benit P. The complete DNA sequence of yeast chromosome III. Nature. 1992 May 7;357(6373):38–46. doi: 10.1038/357038a0. [DOI] [PubMed] [Google Scholar]
- Park E. C., Finley D., Szostak J. W. A strategy for the generation of conditional mutations by protein destabilization. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1249–1252. doi: 10.1073/pnas.89.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petitjean A., Bonneaud N., Lacroute F. The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins. Mol Cell Biol. 1995 Sep;15(9):5071–5081. doi: 10.1128/mcb.15.9.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Scheer U., Thiry M., Goessens G. Structure, function and assembly of the nucleolus. Trends Cell Biol. 1993 Jul;3(7):236–241. doi: 10.1016/0962-8924(93)90123-i. [DOI] [PubMed] [Google Scholar]
- Stettler S., Chiannilkulchai N., Hermann-Le Denmat S., Lalo D., Lacroute F., Sentenac A., Thuriaux P. A general suppressor of RNA polymerase I, II and III mutations in Saccharomyces cerevisiae. Mol Gen Genet. 1993 May;239(1-2):169–176. doi: 10.1007/BF00281615. [DOI] [PubMed] [Google Scholar]
- Sun C., Woolford J. L., Jr The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits. EMBO J. 1994 Jul 1;13(13):3127–3135. doi: 10.1002/j.1460-2075.1994.tb06611.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]