Skip to main content
RNA logoLink to RNA
. 2002 May;8(5):626–636. doi: 10.1017/s1355838202020022

Yeast Pescadillo is required for multiple activities during 60S ribosomal subunit synthesis.

Marlene Oeffinger 1, Anthony Leung 1, Angus Lamond 1, David Tollervey 1, Anthony Lueng 1
PMCID: PMC1370283  PMID: 12022229

Abstract

The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression. Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus. Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rpl25p, consistent with a role in subunit assembly. Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5.8S(L) and 5.8Ss. In cells depleted for Nop7p, the 27SA3 pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8Ss rRNA were depleted. Less depletion was seen for the 5.8S(L) pathway. TAP-tagged Nop7p coprecipitated precursors to both 5.8S(L) and 5.8Ss but not the mature rRNAs. We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA3 pre-rRNA and has additional functions in 60S subunit assembly and transport. Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. 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]
  2. Allmang C., Kufel J., Chanfreau G., Mitchell P., Petfalski E., Tollervey D. Functions of the exosome in rRNA, snoRNA and snRNA synthesis. EMBO J. 1999 Oct 1;18(19):5399–5410. doi: 10.1093/emboj/18.19.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bassler J., Grandi P., Gadal O., Lessmann T., Petfalski E., Tollervey D., Lechner J., Hurt E. Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol Cell. 2001 Sep;8(3):517–529. doi: 10.1016/s1097-2765(01)00342-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Biggiogera M., Bürki K., Kaufmann S. H., Shaper J. H., Gas N., Amalric F., Fakan S. Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy. Development. 1990 Dec;110(4):1263–1270. doi: 10.1242/dev.110.4.1263. [DOI] [PubMed] [Google Scholar]
  7. Bork P., Hofmann K., Bucher P., Neuwald A. F., Altschul S. F., Koonin E. V. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 1997 Jan;11(1):68–76. [PubMed] [Google Scholar]
  8. Ding D. Q., Tomita Y., Yamamoto A., Chikashige Y., Haraguchi T., Hiraoka Y. Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library. Genes Cells. 2000 Mar;5(3):169–190. doi: 10.1046/j.1365-2443.2000.00317.x. [DOI] [PubMed] [Google Scholar]
  9. Fatica Alessandro, Cronshaw Andrew D., Dlakić Mensur, Tollervey David. Ssf1p prevents premature processing of an early pre-60S ribosomal particle. Mol Cell. 2002 Feb;9(2):341–351. doi: 10.1016/s1097-2765(02)00458-6. [DOI] [PubMed] [Google Scholar]
  10. Fuchs S. Y., Adler V., Buschmann T., Wu X., Ronai Z. Mdm2 association with p53 targets its ubiquitination. Oncogene. 1998 Nov 12;17(19):2543–2547. doi: 10.1038/sj.onc.1202200. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gadal O., Strauss D., Kessl J., Trumpower B., Tollervey D., Hurt E. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol Cell Biol. 2001 May;21(10):3405–3415. doi: 10.1128/MCB.21.10.3405-3415.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geerlings T. H., Vos J. C., Raué H. A. The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases. RNA. 2000 Dec;6(12):1698–1703. doi: 10.1017/s1355838200001540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geyer R. K., Yu Z. K., Maki C. G. The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat Cell Biol. 2000 Sep;2(9):569–573. doi: 10.1038/35023507. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Haque J., Boger S., Li J., Duncan S. A. The murine Pes1 gene encodes a nuclear protein containing a BRCT domain. Genomics. 2000 Dec 1;70(2):201–210. doi: 10.1006/geno.2000.6375. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Ho J. H., Kallstrom G., Johnson A. W. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J Cell Biol. 2000 Nov 27;151(5):1057–1066. doi: 10.1083/jcb.151.5.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hurt E., Hannus S., Schmelzl B., Lau D., Tollervey D., Simos G. A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants. J Cell Biol. 1999 Feb 8;144(3):389–401. doi: 10.1083/jcb.144.3.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kinoshita Y., Jarell A. D., Flaman J. M., Foltz G., Schuster J., Sopher B. L., Irvin D. K., Kanning K., Kornblum H. I., Nelson P. S. Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells. J Biol Chem. 2000 Nov 8;276(9):6656–6665. doi: 10.1074/jbc.M008536200. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lafontaine D. L., Tollervey D. The function and synthesis of ribosomes. Nat Rev Mol Cell Biol. 2001 Jul;2(7):514–520. doi: 10.1038/35080045. [DOI] [PubMed] [Google Scholar]
  24. Lafontaine D., Tollervey D. One-step PCR mediated strategy for the construction of conditionally expressed and epitope tagged yeast proteins. Nucleic Acids Res. 1996 Sep 1;24(17):3469–3471. doi: 10.1093/nar/24.17.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Léger-Silvestre I., Noaillac-Depeyre J., Faubladier M., Gas N. Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe. Eur J Cell Biol. 1997 Jan;72(1):13–23. [PubMed] [Google Scholar]
  26. Léger-Silvestre I., Trumtel S., Noaillac-Depeyre J., Gas N. Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae. Chromosoma. 1999 May;108(2):103–113. doi: 10.1007/s004120050357. [DOI] [PubMed] [Google Scholar]
  27. Milkereit P., Gadal O., Podtelejnikov A., Trumtel S., Gas N., Petfalski E., Tollervey D., Mann M., Hurt E., Tschochner H. Maturation and intranuclear transport of pre-ribosomes requires Noc proteins. Cell. 2001 May 18;105(4):499–509. doi: 10.1016/s0092-8674(01)00358-0. [DOI] [PubMed] [Google Scholar]
  28. Rappsilber Juri, Ryder Ursula, Lamond Angus I., Mann Matthias. Large-scale proteomic analysis of the human spliceosome. Genome Res. 2002 Aug;12(8):1231–1245. doi: 10.1101/gr.473902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rigaut G., Shevchenko A., Rutz B., Wilm M., Mann M., Séraphin B. A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol. 1999 Oct;17(10):1030–1032. doi: 10.1038/13732. [DOI] [PubMed] [Google Scholar]
  30. Sakumoto N., Yamashita H., Mukai Y., Kaneko Y., Harashima S. Dual-specificity protein phosphatase Yvh1p, which is required for vegetative growth and sporulation, interacts with yeast pescadillo homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 2001 Nov 30;289(2):608–615. doi: 10.1006/bbrc.2001.6021. [DOI] [PubMed] [Google Scholar]
  31. Savkur R. S., Olson M. O. Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease. Nucleic Acids Res. 1998 Oct 1;26(19):4508–4515. doi: 10.1093/nar/26.19.4508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scheer U., Hock R. Structure and function of the nucleolus. Curr Opin Cell Biol. 1999 Jun;11(3):385–390. doi: 10.1016/S0955-0674(99)80054-4. [DOI] [PubMed] [Google Scholar]
  33. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shaw P. J., Jordan E. G. The nucleolus. Annu Rev Cell Dev Biol. 1995;11:93–121. doi: 10.1146/annurev.cb.11.110195.000521. [DOI] [PubMed] [Google Scholar]
  36. Shou W., Sakamoto K. M., Keener J., Morimoto K. W., Traverso E. E., Azzam R., Hoppe G. J., Feldman R. M., DeModena J., Moazed D. Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit. Mol Cell. 2001 Jul;8(1):45–55. doi: 10.1016/s1097-2765(01)00291-x. [DOI] [PubMed] [Google Scholar]
  37. Shou W., Seol J. H., Shevchenko A., Baskerville C., Moazed D., Chen Z. W., Jang J., Shevchenko A., Charbonneau H., Deshaies R. J. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell. 1999 Apr 16;97(2):233–244. doi: 10.1016/s0092-8674(00)80733-3. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Tao W., Levine A. J. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6937–6941. doi: 10.1073/pnas.96.12.6937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Tollervey D., Lehtonen H., Jansen R., Kern H., Hurt E. C. Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell. 1993 Feb 12;72(3):443–457. doi: 10.1016/0092-8674(93)90120-f. [DOI] [PubMed] [Google Scholar]
  42. Tollervey D., Mattaj I. W. Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J. 1987 Feb;6(2):469–476. doi: 10.1002/j.1460-2075.1987.tb04777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Warner J. R. Nascent ribosomes. Cell. 2001 Oct 19;107(2):133–136. doi: 10.1016/s0092-8674(01)00531-1. [DOI] [PubMed] [Google Scholar]
  45. Weber J. D., Taylor L. J., Roussel M. F., Sherr C. J., Bar-Sagi D. Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol. 1999 May;1(1):20–26. doi: 10.1038/8991. [DOI] [PubMed] [Google Scholar]
  46. Zhang Y., Xiong Y. Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol Cell. 1999 May;3(5):579–591. doi: 10.1016/s1097-2765(00)80351-2. [DOI] [PubMed] [Google Scholar]
  47. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES