Abstract
To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.
Full Text
The Full Text of this article is available as a PDF (3.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Allende M. L., Amsterdam A., Becker T., Kawakami K., Gaiano N., Hopkins N. Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development. Genes Dev. 1996 Dec 15;10(24):3141–3155. doi: 10.1101/gad.10.24.3141. [DOI] [PubMed] [Google Scholar]
- Anderson S. F., Schlegel B. P., Nakajima T., Wolpin E. S., Parvin J. D. BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A. Nat Genet. 1998 Jul;19(3):254–256. doi: 10.1038/930. [DOI] [PubMed] [Google Scholar]
- BROWN D. D., GURDON J. B. ABSENCE OF RIBOSOMAL RNA SYNTHESIS IN THE ANUCLEOLATE MUTANT OF XENOPUS LAEVIS. Proc Natl Acad Sci U S A. 1964 Jan;51:139–146. doi: 10.1073/pnas.51.1.139. [DOI] [PMC free article] [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]
- 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]
- Boncinelli E., Graziani F., Polito L., Malva C., Ritossa F. rDNA magnification at the bobbed locus of the Y chromosome in Drosophila melanogaster. Cell Differ. 1972 Jun;1(2):133–142. doi: 10.1016/0045-6039(72)90036-x. [DOI] [PubMed] [Google Scholar]
- Brown D. D., Weber C. S. Gene linkage by RNA-DNA hybridization. I. Unique DNA sequences homologous to 4 s RNA, 5 s RNA and ribosomal RNA. J Mol Biol. 1968 Jun 28;34(3):661–680. doi: 10.1016/0022-2836(68)90188-5. [DOI] [PubMed] [Google Scholar]
- Burger F., Daugeron M. C., Linder P. Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis. Nucleic Acids Res. 2000 Jun 15;28(12):2315–2323. doi: 10.1093/nar/28.12.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Caizergues-Ferrer M., Mathieu C., Mariottini P., Amalric F., Amaldi F. Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis. Development. 1991 May;112(1):317–326. doi: 10.1242/dev.112.1.317. [DOI] [PubMed] [Google Scholar]
- Callebaut I., Mornon J. P. From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair. FEBS Lett. 1997 Jan 2;400(1):25–30. doi: 10.1016/s0014-5793(96)01312-9. [DOI] [PubMed] [Google Scholar]
- Daugeron M. C., Linder P. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly. RNA. 1998 May;4(5):566–581. doi: 10.1017/s1355838298980190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deshmukh M., Stark J., Yeh L. C., Lee J. C., Woolford J. L., Jr Multiple regions of yeast ribosomal protein L1 are important for its interaction with 5 S rRNA and assembly into ribosomes. J Biol Chem. 1995 Dec 15;270(50):30148–30156. doi: 10.1074/jbc.270.50.30148. [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]
- Doye V., Hurt E. C. Genetic approaches to nuclear pore structure and function. Trends Genet. 1995 Jun;11(6):235–241. doi: 10.1016/s0168-9525(00)89057-5. [DOI] [PubMed] [Google Scholar]
- ELSDALE T. R., FISCHBERG M., SMITH S. A mutation that reduces nucleolar number in Xenopus laevis. Exp Cell Res. 1958 Jun;14(3):642–643. doi: 10.1016/0014-4827(58)90175-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Fabre E., Boelens W. C., Wimmer C., Mattaj I. W., Hurt E. C. Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif. Cell. 1994 Jul 29;78(2):275–289. doi: 10.1016/0092-8674(94)90297-6. [DOI] [PubMed] [Google Scholar]
- Frank D., Patterson B., Guthrie C. Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing. Mol Cell Biol. 1992 Nov;12(11):5197–5205. doi: 10.1128/mcb.12.11.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gadal O., Strauss D., Braspenning J., Hoepfner D., Petfalski E., Philippsen P., Tollervey D., Hurt E. A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits. EMBO J. 2001 Jul 16;20(14):3695–3704. doi: 10.1093/emboj/20.14.3695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gaiano N., Amsterdam A., Kawakami K., Allende M., Becker T., Hopkins N. Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature. 1996 Oct 31;383(6603):829–832. doi: 10.1038/383829a0. [DOI] [PubMed] [Google Scholar]
- Giordano E., Peluso I., Senger S., Furia M. minifly, a Drosophila gene required for ribosome biogenesis. J Cell Biol. 1999 Mar 22;144(6):1123–1133. doi: 10.1083/jcb.144.6.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harnpicharnchai P., Jakovljevic J., Horsey E., Miles T., Roman J., Rout M., Meagher D., Imai B., Guo Y., Brame C. J. Composition and functional characterization of yeast 66S ribosome assembly intermediates. Mol Cell. 2001 Sep;8(3):505–515. doi: 10.1016/s1097-2765(01)00344-6. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Hong B., Brockenbrough J. S., Wu P., Aris J. P. Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast. Mol Cell Biol. 1997 Jan;17(1):378–388. doi: 10.1128/mcb.17.1.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Kay M. A., Jacobs-Lorena M. Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster. Mol Cell Biol. 1985 Dec;5(12):3583–3592. doi: 10.1128/mcb.5.12.3583. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koonin E. V., Altschul S. F., Bork P. BRCA1 protein products ... Functional motifs... Nat Genet. 1996 Jul;13(3):266–268. doi: 10.1038/ng0796-266. [DOI] [PubMed] [Google Scholar]
- Kressler D., Linder P., de La Cruz J. Protein trans-acting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Dec;19(12):7897–7912. doi: 10.1128/mcb.19.12.7897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kressler D., de la Cruz J., Rojo M., Linder P. Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae. Mol Cell Biol. 1998 Apr;18(4):1855–1865. doi: 10.1128/mcb.18.4.1855. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liao X. C., Tang J., Rosbash M. An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing. Genes Dev. 1993 Mar;7(3):419–428. doi: 10.1101/gad.7.3.419. [DOI] [PubMed] [Google Scholar]
- Margossian S. P., Li H., Zassenhaus H. P., Butow R. A. The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity. Cell. 1996 Jan 26;84(2):199–209. doi: 10.1016/s0092-8674(00)80975-7. [DOI] [PubMed] [Google Scholar]
- Mitchell P., Petfalski E., Shevchenko A., Mann M., Tollervey D. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases. Cell. 1997 Nov 14;91(4):457–466. doi: 10.1016/s0092-8674(00)80432-8. [DOI] [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]
- Pause A., Méthot N., Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. doi: 10.1128/mcb.13.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierandrei-Amaldi P., Beccari E., Bozzoni I., Amaldi F. Ribosomal protein production in normal and anucleolate Xenopus embryos: regulation at the posttranscriptional and translational levels. Cell. 1985 Aug;42(1):317–323. doi: 10.1016/s0092-8674(85)80127-6. [DOI] [PubMed] [Google Scholar]
- Pierandrei-Amaldi P., Campioni N., Beccari E., Bozzoni I., Amaldi F. Expression of ribosomal-protein genes in Xenopus laevis development. Cell. 1982 Aug;30(1):163–171. doi: 10.1016/0092-8674(82)90022-8. [DOI] [PubMed] [Google Scholar]
- Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
- Procunier J. D., Tartof K. D. Genetic analysis of the 5S RNA genes in Drosophila melanogaster. Genetics. 1975 Nov;81(3):515–523. doi: 10.1093/genetics/81.3.515. [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]
- Ripmaster T. L., Vaughn G. P., Woolford J. L., Jr DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7901–7912. doi: 10.1128/mcb.13.12.7901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Schwer B. A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat Struct Biol. 2001 Feb;8(2):113–116. doi: 10.1038/84091. [DOI] [PubMed] [Google Scholar]
- Simos G., Segref A., Fasiolo F., Hellmuth K., Shevchenko A., Mann M., Hurt E. C. The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. EMBO J. 1996 Oct 1;15(19):5437–5448. [PMC free article] [PubMed] [Google Scholar]
- Stage-Zimmermann T., Schmidt U., Silver P. A. Factors affecting nuclear export of the 60S ribosomal subunit in vivo. Mol Biol Cell. 2000 Nov;11(11):3777–3789. doi: 10.1091/mbc.11.11.3777. [DOI] [PMC free article] [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]
- Tanner N. K., Linder P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell. 2001 Aug;8(2):251–262. doi: 10.1016/s1097-2765(01)00329-x. [DOI] [PubMed] [Google Scholar]
- Venema J., Bousquet-Antonelli C., Gelugne J. P., Caizergues-Ferrer M., Tollervey D. Rok1p is a putative RNA helicase required for rRNA processing. Mol Cell Biol. 1997 Jun;17(6):3398–3407. doi: 10.1128/mcb.17.6.3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Venema J., Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae. Annu Rev Genet. 1999;33:261–311. doi: 10.1146/annurev.genet.33.1.261. [DOI] [PubMed] [Google Scholar]
- Vilardell J., Warner J. R. Ribosomal protein L32 of Saccharomyces cerevisiae influences both the splicing of its own transcript and the processing of rRNA. Mol Cell Biol. 1997 Apr;17(4):1959–1965. doi: 10.1128/mcb.17.4.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace H., Birnstiel M. L. Ribosomal cistrons and the nucleolar organizer. Biochim Biophys Acta. 1966 Feb 21;114(2):296–310. doi: 10.1016/0005-2787(66)90311-x. [DOI] [PubMed] [Google Scholar]
- Warner J. R. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999 Nov;24(11):437–440. doi: 10.1016/s0968-0004(99)01460-7. [DOI] [PubMed] [Google Scholar]
- Weaver P. L., Sun C., Chang T. H. Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3. Mol Cell Biol. 1997 Mar;17(3):1354–1365. doi: 10.1128/mcb.17.3.1354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu D., Field D. J., Tang S. J., Moris A., Bobechko B. P., Friesen J. D. Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing. Mol Cell Biol. 1998 Apr;18(4):2055–2066. doi: 10.1128/mcb.18.4.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zanchin N. I., Roberts P., DeSilva A., Sherman F., Goldfarb D. S. Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis. Mol Cell Biol. 1997 Sep;17(9):5001–5015. doi: 10.1128/mcb.17.9.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang X., Moréra S., Bates P. A., Whitehead P. C., Coffer A. I., Hainbucher K., Nash R. A., Sternberg M. J., Lindahl T., Freemont P. S. Structure of an XRCC1 BRCT domain: a new protein-protein interaction module. EMBO J. 1998 Nov 2;17(21):6404–6411. doi: 10.1093/emboj/17.21.6404. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de la Cruz J., Kressler D., Linder P. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci. 1999 May;24(5):192–198. doi: 10.1016/s0968-0004(99)01376-6. [DOI] [PubMed] [Google Scholar]
- de la Cruz J., Kressler D., Rojo M., Tollervey D., Linder P. Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae. RNA. 1998 Oct;4(10):1268–1281. doi: 10.1017/s1355838298981158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de la Cruz J., Kressler D., Tollervey D., Linder P. Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3' end formation of 5.8S rRNA in Saccharomyces cerevisiae. EMBO J. 1998 Feb 16;17(4):1128–1140. doi: 10.1093/emboj/17.4.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]