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. 1996 May 1;15(9):2270–2284.

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

G Simos 1, H Tekotte 1, H Grosjean 1, A Segref 1, K Sharma 1, D Tollervey 1, E C Hurt 1
PMCID: PMC450152  PMID: 8641292

Abstract

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

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Selected References

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

  1. Baim S. B., Pietras D. F., Eustice D. C., Sherman F. A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c. Mol Cell Biol. 1985 Aug;5(8):1839–1846. doi: 10.1128/mcb.5.8.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becker J., Melchior F., Gerke V., Bischoff F. R., Ponstingl H., Wittinghofer A. RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae. J Biol Chem. 1995 May 19;270(20):11860–11865. doi: 10.1074/jbc.270.20.11860. [DOI] [PubMed] [Google Scholar]
  3. Bischoff F. R., Krebber H., Kempf T., Hermes I., Ponstingl H. Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1749–1753. doi: 10.1073/pnas.92.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng Y., Dahlberg J. E., Lund E. Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport. Science. 1995 Mar 24;267(5205):1807–1810. doi: 10.1126/science.7534442. [DOI] [PubMed] [Google Scholar]
  5. Clark M. W., Abelson J. The subnuclear localization of tRNA ligase in yeast. J Cell Biol. 1987 Oct;105(4):1515–1526. doi: 10.1083/jcb.105.4.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Corbett A. H., Koepp D. M., Schlenstedt G., Lee M. S., Hopper A. K., Silver P. A. Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import. J Cell Biol. 1995 Sep;130(5):1017–1026. doi: 10.1083/jcb.130.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Culbertson M. R., Winey M. Split tRNA genes and their products: a paradigm for the study of cell function and evolution. Yeast. 1989 Nov-Dec;5(6):405–427. doi: 10.1002/yea.320050602. [DOI] [PubMed] [Google Scholar]
  8. Davis L. I. The nuclear pore complex. Annu Rev Biochem. 1995;64:865–896. doi: 10.1146/annurev.bi.64.070195.004245. [DOI] [PubMed] [Google Scholar]
  9. Doye V., Wepf R., Hurt E. C. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. doi: 10.1002/j.1460-2075.1994.tb06953.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  13. Gerace L. Nuclear export signals and the fast track to the cytoplasm. Cell. 1995 Aug 11;82(3):341–344. doi: 10.1016/0092-8674(95)90420-4. [DOI] [PubMed] [Google Scholar]
  14. Gorsch L. C., Dockendorff T. C., Cole C. N. A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes. J Cell Biol. 1995 May;129(4):939–955. doi: 10.1083/jcb.129.4.939. [DOI] [PMC free article] [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. Grandi P., Emig S., Weise C., Hucho F., Pohl T., Hurt E. C. A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. J Cell Biol. 1995 Sep;130(6):1263–1273. doi: 10.1083/jcb.130.6.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grandi P., Schlaich N., Tekotte H., Hurt E. C. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. EMBO J. 1995 Jan 3;14(1):76–87. doi: 10.1002/j.1460-2075.1995.tb06977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grosjean H., Edqvist J., Stråby K. B., Giegé R. Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture. J Mol Biol. 1996 Jan 12;255(1):67–85. doi: 10.1006/jmbi.1996.0007. [DOI] [PubMed] [Google Scholar]
  19. Grosjean H., Sprinzl M., Steinberg S. Posttranscriptionally modified nucleosides in transfer RNA: their locations and frequencies. Biochimie. 1995;77(1-2):139–141. doi: 10.1016/0300-9084(96)88117-x. [DOI] [PubMed] [Google Scholar]
  20. Haselbeck R. C., Greer C. L. Minimum intron requirements for tRNA splicing and nuclear transport in Xenopus oocytes. Biochemistry. 1993 Aug 24;32(33):8575–8581. doi: 10.1021/bi00084a026. [DOI] [PubMed] [Google Scholar]
  21. Hopper A. K., Banks F. A yeast mutant which accumulates precursor tRNAs. Cell. 1978 Jun;14(2):211–219. doi: 10.1016/0092-8674(78)90108-3. [DOI] [PubMed] [Google Scholar]
  22. Hopper A. K. Genetic methods for study of trans-acting genes involved in processing of precursors to yeast cytoplasmic transfer RNAs. Methods Enzymol. 1990;181:400–421. doi: 10.1016/0076-6879(90)81139-l. [DOI] [PubMed] [Google Scholar]
  23. Hopper A. K., Schultz L. D., Shapiro R. A. Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs. Cell. 1980 Mar;19(3):741–751. doi: 10.1016/s0092-8674(80)80050-x. [DOI] [PubMed] [Google Scholar]
  24. Hopper A. K., Traglia H. M., Dunst R. W. The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J Cell Biol. 1990 Aug;111(2):309–321. doi: 10.1083/jcb.111.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hurt D. J., Wang S. S., Lin Y. H., Hopper A. K. Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing. Mol Cell Biol. 1987 Mar;7(3):1208–1216. doi: 10.1128/mcb.7.3.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hurt E. C., McDowall A., Schimmang T. Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae. Eur J Cell Biol. 1988 Aug;46(3):554–563. [PubMed] [Google Scholar]
  27. Izaurralde E., Mattaj I. W. RNA export. Cell. 1995 Apr 21;81(2):153–159. doi: 10.1016/0092-8674(95)90323-2. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Jarmolowski A., Boelens W. C., Izaurralde E., Mattaj I. W. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. doi: 10.1083/jcb.124.5.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Johnson P. F., Abelson J. The yeast tRNATyr gene intron is essential for correct modification of its tRNA product. Nature. 1983 Apr 21;302(5910):681–687. doi: 10.1038/302681a0. [DOI] [PubMed] [Google Scholar]
  31. Kammen H. O., Marvel C. C., Hardy L., Penhoet E. E. Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. J Biol Chem. 1988 Feb 15;263(5):2255–2263. [PubMed] [Google Scholar]
  32. 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]
  33. Li O., Heath C. V., Amberg D. C., Dockendorff T. C., Copeland C. S., Snyder M., Cole C. N. Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes. Mol Biol Cell. 1995 Apr;6(4):401–417. doi: 10.1091/mbc.6.4.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lim F., Morris C. P., Occhiodoro F., Wallace J. C. Sequence and domain structure of yeast pyruvate carboxylase. J Biol Chem. 1988 Aug 15;263(23):11493–11497. [PubMed] [Google Scholar]
  35. Marck C., Lefebvre O., Carles C., Riva M., Chaussivert N., Ruet A., Sentenac A. The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4027–4031. doi: 10.1073/pnas.90.9.4027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  37. Nehrbass U., Fabre E., Dihlmann S., Herth W., Hurt E. C. Analysis of nucleo-cytoplasmic transport in a thermosensitive mutant of nuclear pore protein NSP1. Eur J Cell Biol. 1993 Oct;62(1):1–12. [PubMed] [Google Scholar]
  38. Nehrbass U., Kern H., Mutvei A., Horstmann H., Marshallsay B., Hurt E. C. NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell. 1990 Jun 15;61(6):979–989. doi: 10.1016/0092-8674(90)90063-k. [DOI] [PubMed] [Google Scholar]
  39. Nishikura K., De Robertis E. M. RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. J Mol Biol. 1981 Jan 15;145(2):405–420. doi: 10.1016/0022-2836(81)90212-6. [DOI] [PubMed] [Google Scholar]
  40. Nurse K., Wrzesinski J., Bakin A., Lane B. G., Ofengand J. Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. RNA. 1995 Mar;1(1):102–112. [PMC free article] [PubMed] [Google Scholar]
  41. O'Connor J. P., Peebles C. L. PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing. Mol Cell Biol. 1992 Sep;12(9):3843–3856. doi: 10.1128/mcb.12.9.3843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  43. Peebles C. L., Ogden R. C., Knapp G., Abelson J. Splicing of yeast tRNA precursors: a two-stage reaction. Cell. 1979 Sep;18(1):27–35. doi: 10.1016/0092-8674(79)90350-7. [DOI] [PubMed] [Google Scholar]
  44. Rose A. M., Belford H. G., Shen W. C., Greer C. L., Hopper A. K., Martin N. C. Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase. Biochimie. 1995;77(1-2):45–53. doi: 10.1016/0300-9084(96)88103-x. [DOI] [PubMed] [Google Scholar]
  45. Samuelsson T., Olsson M. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae. J Biol Chem. 1990 May 25;265(15):8782–8787. [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schlaich N. L., Hurt E. C. Analysis of nucleocytoplasmic transport and nuclear envelope structure in yeast disrupted for the gene encoding the nuclear pore protein Nup1p. Eur J Cell Biol. 1995 May;67(1):8–14. [PubMed] [Google Scholar]
  48. Sharma K., Fabre E., Tekotte H., Hurt E. C., Tollervey D. Yeast nucleoporin mutants are defective in pre-tRNA splicing. Mol Cell Biol. 1996 Jan;16(1):294–301. doi: 10.1128/mcb.16.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shen W. C., Selvakumar D., Stanford D. R., Hopper A. K. The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix. J Biol Chem. 1993 Sep 15;268(26):19436–19444. [PubMed] [Google Scholar]
  50. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  51. Simos G., Hurt E. C. Nucleocytoplasmic transport: factors and mechanisms. FEBS Lett. 1995 Aug 1;369(1):107–112. doi: 10.1016/0014-5793(95)00674-x. [DOI] [PubMed] [Google Scholar]
  52. Siniossoglou S., Wimmer C., Rieger M., Doye V., Tekotte H., Weise C., Emig S., Segref A., Hurt E. C. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell. 1996 Jan 26;84(2):265–275. doi: 10.1016/s0092-8674(00)80981-2. [DOI] [PubMed] [Google Scholar]
  53. Steinberg S., Misch A., Sprinzl M. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1993 Jul 1;21(13):3011–3015. doi: 10.1093/nar/21.13.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Strobel M. C., Abelson J. Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA. Mol Cell Biol. 1986 Jul;6(7):2674–2683. doi: 10.1128/mcb.6.7.2674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Szweykowska-Kulinska Z., Senger B., Keith G., Fasiolo F., Grosjean H. Intron-dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile). EMBO J. 1994 Oct 3;13(19):4636–4644. doi: 10.1002/j.1460-2075.1994.tb06786.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tobian J. A., Drinkard L., Zasloff M. tRNA nuclear transport: defining the critical regions of human tRNAimet by point mutagenesis. Cell. 1985 Dec;43(2 Pt 1):415–422. doi: 10.1016/0092-8674(85)90171-0. [DOI] [PubMed] [Google Scholar]
  57. Tranguch A. J., Engelke D. R. Comparative structural analysis of nuclear RNase P RNAs from yeast. J Biol Chem. 1993 Jul 5;268(19):14045–14055. [PubMed] [Google Scholar]
  58. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  59. Wimmer C., Doye V., Grandi P., Nehrbass U., Hurt E. C. A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1. EMBO J. 1992 Dec;11(13):5051–5061. doi: 10.1002/j.1460-2075.1992.tb05612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wrzesinski J., Bakin A., Nurse K., Lane B. G., Ofengand J. Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli. Biochemistry. 1995 Jul 11;34(27):8904–8913. doi: 10.1021/bi00027a043. [DOI] [PubMed] [Google Scholar]
  61. Wrzesinski J., Nurse K., Bakin A., Lane B. G., Ofengand J. A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe). RNA. 1995 Jun;1(4):437–448. [PMC free article] [PubMed] [Google Scholar]
  62. Zasloff M. tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6436–6440. doi: 10.1073/pnas.80.21.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]

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