Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Nov;16(11):6046–6054. doi: 10.1128/mcb.16.11.6046

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Y Huang 1, G G Carmichael 1
PMCID: PMC231607  PMID: 8887634

Abstract

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Adami G. R., Carmichael G. G. The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability. Nucleic Acids Res. 1987 Mar 25;15(6):2593–2610. doi: 10.1093/nar/15.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adami G. R., Marlor C. W., Barrett N. L., Carmichael G. G. Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs. J Virol. 1989 Jan;63(1):85–93. doi: 10.1128/jvi.63.1.85-93.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alonso-Caplen F. V., Nemeroff M. E., Qiu Y., Krug R. M. Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev. 1992 Feb;6(2):255–267. doi: 10.1101/gad.6.2.255. [DOI] [PubMed] [Google Scholar]
  4. Barrett N. L., Carmichael G. G., Luo Y. Splice site requirement for the efficient accumulation of polyoma virus late mRNAs. Nucleic Acids Res. 1991 Jun 11;19(11):3011–3017. doi: 10.1093/nar/19.11.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrett N. L., Li X., Carmichael G. G. The sequence and context of the 5' splice site govern the nuclear stability of polyoma virus late RNAs. Nucleic Acids Res. 1995 Dec 11;23(23):4812–4817. doi: 10.1093/nar/23.23.4812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Batt D. B., Rapp L. M., Carmichael G. G. Splice site selection in polyomavirus late pre-mRNA processing. J Virol. 1994 Mar;68(3):1797–1804. doi: 10.1128/jvi.68.3.1797-1804.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bray M., Prasad S., Dubay J. W., Hunter E., Jeang K. T., Rekosh D., Hammarskjöld M. L. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1256–1260. doi: 10.1073/pnas.91.4.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cahill K. B., Carmichael G. G. Deletion analysis of the polyomavirus late promoter: evidence for both positive and negative elements in the absence of early proteins. J Virol. 1989 Sep;63(9):3634–3642. doi: 10.1128/jvi.63.9.3634-3642.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang D. D., Sharp P. A. Regulation by HIV Rev depends upon recognition of splice sites. Cell. 1989 Dec 1;59(5):789–795. doi: 10.1016/0092-8674(89)90602-8. [DOI] [PubMed] [Google Scholar]
  11. Cullen B. R. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol Rev. 1992 Sep;56(3):375–394. doi: 10.1128/mr.56.3.375-394.1992. [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. Fischer U., Meyer S., Teufel M., Heckel C., Lührmann R., Rautmann G. Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO J. 1994 Sep 1;13(17):4105–4112. doi: 10.1002/j.1460-2075.1994.tb06728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  15. Green M. R. Pre-mRNA processing and mRNA nuclear export. Curr Opin Cell Biol. 1989 Jun;1(3):519–525. doi: 10.1016/0955-0674(89)90014-8. [DOI] [PubMed] [Google Scholar]
  16. Gruss P., Lai C. J., Dhar R., Khoury G. Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4317–4321. doi: 10.1073/pnas.76.9.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamer D. H., Smith K. D., Boyer S. H., Leder P. SV40 recombinants carrying rabbit beta-globin gene coding sequences. Cell. 1979 Jul;17(3):725–735. doi: 10.1016/0092-8674(79)90279-4. [DOI] [PubMed] [Google Scholar]
  18. Huang J., Liang T. J. A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products. Mol Cell Biol. 1993 Dec;13(12):7476–7486. doi: 10.1128/mcb.13.12.7476. [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. Huang Z. M., Yen T. S. Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Mol Cell Biol. 1995 Jul;15(7):3864–3869. doi: 10.1128/mcb.15.7.3864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hyde-DeRuyscher R. P., Carmichael G. G. Polyomavirus late pre-mRNA processing: DNA replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch. J Virol. 1990 Dec;64(12):5823–5832. doi: 10.1128/jvi.64.12.5823-5832.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kalland K. H., Szilvay A. M., Langhoff E., Haukenes G. Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm. J Virol. 1994 Mar;68(3):1475–1485. doi: 10.1128/jvi.68.3.1475-1485.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Katz R. A., Skalka A. M. Control of retroviral RNA splicing through maintenance of suboptimal processing signals. Mol Cell Biol. 1990 Feb;10(2):696–704. doi: 10.1128/mcb.10.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kjems J., Sharp P. A. The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J Virol. 1993 Aug;67(8):4769–4776. doi: 10.1128/jvi.67.8.4769-4776.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krug R. M. The regulation of export of mRNA from nucleus to cytoplasm. Curr Opin Cell Biol. 1993 Dec;5(6):944–949. doi: 10.1016/0955-0674(93)90074-z. [DOI] [PubMed] [Google Scholar]
  27. Legrain P., Rosbash M. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell. 1989 May 19;57(4):573–583. doi: 10.1016/0092-8674(89)90127-x. [DOI] [PubMed] [Google Scholar]
  28. Lichtler A., Barrett N. L., Carmichael G. G. Simple, inexpensive preparation of T1/T2 ribonuclease suitable for use in RNase protection experiments. Biotechniques. 1992 Feb;12(2):231–232. [PubMed] [Google Scholar]
  29. Liu X., Mertz J. E. HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression. Genes Dev. 1995 Jul 15;9(14):1766–1780. doi: 10.1101/gad.9.14.1766. [DOI] [PubMed] [Google Scholar]
  30. Lu X. B., Heimer J., Rekosh D., Hammarskjöld M. L. U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7598–7602. doi: 10.1073/pnas.87.19.7598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luo Y., Carmichael G. G. Splice site choice in a complex transcription unit containing multiple inefficient polyadenylation signals. Mol Cell Biol. 1991 Oct;11(10):5291–5300. doi: 10.1128/mcb.11.10.5291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Malim M. H., Cullen B. R. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993 Oct;13(10):6180–6189. doi: 10.1128/mcb.13.10.6180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  34. Mattaj I. W. Splicing stories and poly(A) tales: an update on RNA processing and transport. Curr Opin Cell Biol. 1990 Jun;2(3):528–538. doi: 10.1016/0955-0674(90)90138-5. [DOI] [PubMed] [Google Scholar]
  35. McNally M. T., Beemon K. Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing. J Virol. 1992 Jan;66(1):6–11. doi: 10.1128/jvi.66.1.6-11.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meyer B. E., Malim M. H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 1994 Jul 1;8(13):1538–1547. doi: 10.1101/gad.8.13.1538. [DOI] [PubMed] [Google Scholar]
  37. Rosbash M., Singer R. H. RNA travel: tracks from DNA to cytoplasm. Cell. 1993 Nov 5;75(3):399–401. doi: 10.1016/0092-8674(93)90373-x. [DOI] [PubMed] [Google Scholar]
  38. Ruhl M., Himmelspach M., Bahr G. M., Hammerschmid F., Jaksche H., Wolff B., Aschauer H., Farrington G. K., Probst H., Bevec D. Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation. J Cell Biol. 1993 Dec;123(6 Pt 1):1309–1320. doi: 10.1083/jcb.123.6.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ryu W. S., Mertz J. E. Simian virus 40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm. J Virol. 1989 Oct;63(10):4386–4394. doi: 10.1128/jvi.63.10.4386-4394.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  41. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  42. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  43. Stutz F., Neville M., Rosbash M. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 1995 Aug 11;82(3):495–506. doi: 10.1016/0092-8674(95)90438-7. [DOI] [PubMed] [Google Scholar]
  44. Treisman R. Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis. Nucleic Acids Res. 1980 Nov 11;8(21):4867–4888. doi: 10.1093/nar/8.21.4867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zapp M. L. The ins and outs of RNA nucleocytoplasmic transport. Curr Opin Genet Dev. 1995 Apr;5(2):229–233. doi: 10.1016/0959-437x(95)80013-1. [DOI] [PubMed] [Google Scholar]
  46. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES