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. 1997 May;8(5):825–841. doi: 10.1091/mbc.8.5.825

Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.

A K Azad 1, T Tani 1, N Shiki 1, S Tsuneyoshi 1, S Urushiyama 1, Y Ohshima 1
PMCID: PMC276132  PMID: 9168469

Abstract

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.

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Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Bossie M. A., DeHoratius C., Barcelo G., Silver P. A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast. Mol Biol Cell. 1992 Aug;3(8):875–893. doi: 10.1091/mbc.3.8.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown J. A., Bharathi A., Ghosh A., Whalen W., Fitzgerald E., Dhar R. A mutation in the Schizosaccharomyces pombe rae1 gene causes defects in poly(A)+ RNA export and in the cytoskeleton. J Biol Chem. 1995 Mar 31;270(13):7411–7419. doi: 10.1074/jbc.270.13.7411. [DOI] [PubMed] [Google Scholar]
  7. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  8. Cottarel G., Beach D., Deuschle U. Two new multi-purpose multicopy Schizosaccharomyces pombe shuttle vectors, pSP1 and pSP2. Curr Genet. 1993 May-Jun;23(5-6):547–548. doi: 10.1007/BF00312650. [DOI] [PubMed] [Google Scholar]
  9. Dohmen R. J., Stappen R., McGrath J. P., Forrová H., Kolarov J., Goffeau A., Varshavsky A. An essential yeast gene encoding a homolog of ubiquitin-activating enzyme. J Biol Chem. 1995 Jul 28;270(30):18099–18109. doi: 10.1074/jbc.270.30.18099. [DOI] [PubMed] [Google Scholar]
  10. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol. 1985 May;100(5):1706–1715. doi: 10.1083/jcb.100.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eckner R., Ellmeier W., Birnstiel M. L. Mature mRNA 3' end formation stimulates RNA export from the nucleus. EMBO J. 1991 Nov;10(11):3513–3522. doi: 10.1002/j.1460-2075.1991.tb04915.x. [DOI] [PMC free article] [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. Forrester W., Stutz F., Rosbash M., Wickens M. Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure. Genes Dev. 1992 Oct;6(10):1914–1926. doi: 10.1101/gad.6.10.1914. [DOI] [PubMed] [Google Scholar]
  14. French B. T., Schumm D. E., Webb T. E. Active transport of messenger ribonucleoprotein particles in a reconstituted cell-free system. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5163–5166. doi: 10.1073/pnas.84.15.5163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hamm J., Mattaj I. W. Monomethylated cap structures facilitate RNA export from the nucleus. Cell. 1990 Oct 5;63(1):109–118. doi: 10.1016/0092-8674(90)90292-m. [DOI] [PubMed] [Google Scholar]
  17. Heath C. V., Copeland C. S., Amberg D. C., Del Priore V., Snyder M., Cole C. N. Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene. J Cell Biol. 1995 Dec;131(6 Pt 2):1677–1697. doi: 10.1083/jcb.131.6.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirano T., Funahashi S., Uemura T., Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis. EMBO J. 1986 Nov;5(11):2973–2979. doi: 10.1002/j.1460-2075.1986.tb04594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huang Y., Carmichael G. G. Role of polyadenylation in nucleocytoplasmic transport of mRNA. Mol Cell Biol. 1996 Apr;16(4):1534–1542. doi: 10.1128/mcb.16.4.1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Jentsch S., Seufert W., Hauser H. P. Genetic analysis of the ubiquitin system. Biochim Biophys Acta. 1991 Jun 13;1089(2):127–139. doi: 10.1016/0167-4781(91)90001-3. [DOI] [PubMed] [Google Scholar]
  22. Kadowaki T., Chen S., Hitomi M., Jacobs E., Kumagai C., Liang S., Schneiter R., Singleton D., Wisniewska J., Tartakoff A. M. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J Cell Biol. 1994 Aug;126(3):649–659. doi: 10.1083/jcb.126.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kadowaki T., Goldfarb D., Spitz L. M., Tartakoff A. M., Ohno M. Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. EMBO J. 1993 Jul;12(7):2929–2937. doi: 10.1002/j.1460-2075.1993.tb05955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kadowaki T., Hitomi M., Chen S., Tartakoff A. M. Nuclear mRNA accumulation causes nucleolar fragmentation in yeast mtr2 mutant. Mol Biol Cell. 1994 Nov;5(11):1253–1263. doi: 10.1091/mbc.5.11.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Lim C. R., Kimata Y., Oka M., Nomaguchi K., Kohno K. Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. J Biochem. 1995 Jul;118(1):13–17. doi: 10.1093/oxfordjournals.jbchem.a124868. [DOI] [PubMed] [Google Scholar]
  27. Matsumoto T., Beach D. Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase. Cell. 1991 Jul 26;66(2):347–360. doi: 10.1016/0092-8674(91)90624-8. [DOI] [PubMed] [Google Scholar]
  28. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  29. McGrath J. P., Jentsch S., Varshavsky A. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 1991 Jan;10(1):227–236. doi: 10.1002/j.1460-2075.1991.tb07940.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
  31. Moffett R. B., Webb T. E. Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins. Biochemistry. 1981 May 26;20(11):3253–3262. doi: 10.1021/bi00514a042. [DOI] [PubMed] [Google Scholar]
  32. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  33. Nigg E. A., Baeuerle P. A., Lührmann R. Nuclear import-export: in search of signals and mechanisms. Cell. 1991 Jul 12;66(1):15–22. doi: 10.1016/0092-8674(91)90135-l. [DOI] [PubMed] [Google Scholar]
  34. Nischt R., Thüroff E., Küfer N. F. Molecular cloning of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe. Curr Genet. 1986;10(5):365–370. doi: 10.1007/BF00418408. [DOI] [PubMed] [Google Scholar]
  35. Okazaki K., Okazaki N., Kume K., Jinno S., Tanaka K., Okayama H. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res. 1990 Nov 25;18(22):6485–6489. doi: 10.1093/nar/18.22.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  37. Piñol-Roma S., Dreyfuss G. Transcription-dependent and transcription-independent nuclear transport of hnRNP proteins. Science. 1991 Jul 19;253(5017):312–314. doi: 10.1126/science.1857966. [DOI] [PubMed] [Google Scholar]
  38. Santiago T. C., Purvis I. J., Bettany A. J., Brown A. J. The relationship between mRNA stability and length in Saccharomyces cerevisiae. Nucleic Acids Res. 1986 Nov 11;14(21):8347–8360. doi: 10.1093/nar/14.21.8347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sazer S. The search for the primary function of the Ran GTPase continues. Trends Cell Biol. 1996 Mar;6(3):81–85. doi: 10.1016/0962-8924(96)80992-5. [DOI] [PubMed] [Google Scholar]
  40. Schneiter R., Kadowaki T., Tartakoff A. M. mRNA transport in yeast: time to reinvestigate the functions of the nucleolus. Mol Biol Cell. 1995 Apr;6(4):357–370. doi: 10.1091/mbc.6.4.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schröder H. C., Friese U., Bachmann M., Zaubitzer T., Müller W. E. Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei. Eur J Biochem. 1989 Apr 15;181(1):149–158. doi: 10.1111/j.1432-1033.1989.tb14706.x. [DOI] [PubMed] [Google Scholar]
  42. Shiokawa K., Pogo A. O. The role of cytoplasmic membranes in controlling the transport of nuclear messenger RNA and initiation of protein synthesis. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2658–2662. doi: 10.1073/pnas.71.7.2658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Singleton D. R., Chen S., Hitomi M., Kumagai C., Tartakoff A. M. A yeast protein that bidirectionally affects nucleocytoplasmic transport. J Cell Sci. 1995 Jan;108(Pt 1):265–272. doi: 10.1242/jcs.108.1.265. [DOI] [PubMed] [Google Scholar]
  44. Tachibana T., Imamoto N., Seino H., Nishimoto T., Yoneda Y. Loss of RCC1 leads to suppression of nuclear protein import in living cells. J Biol Chem. 1994 Oct 7;269(40):24542–24545. [PubMed] [Google Scholar]
  45. Tani T., Derby R. J., Hiraoka Y., Spector D. L. Nucleolar accumulation of poly (A)+ RNA in heat-shocked yeast cells: implication of nucleolar involvement in mRNA transport. Mol Biol Cell. 1995 Nov;6(11):1515–1534. doi: 10.1091/mbc.6.11.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Uemura T., Tanagida M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 1986 May;5(5):1003–1010. doi: 10.1002/j.1460-2075.1986.tb04315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Urushiyama S., Tani T., Ohshima Y. Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe. Mol Gen Genet. 1996 Nov 27;253(1-2):118–127. doi: 10.1007/s004380050304. [DOI] [PubMed] [Google Scholar]
  49. Uzawa S., Samejima I., Hirano T., Tanaka K., Yanagida M. The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell. 1990 Sep 7;62(5):913–925. doi: 10.1016/0092-8674(90)90266-h. [DOI] [PubMed] [Google Scholar]
  50. Vijayraghavan U., Company M., Abelson J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 1989 Aug;3(8):1206–1216. doi: 10.1101/gad.3.8.1206. [DOI] [PubMed] [Google Scholar]
  51. Zacksenhaus E., Sheinin R. Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication. EMBO J. 1990 Sep;9(9):2923–2929. doi: 10.1002/j.1460-2075.1990.tb07483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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