Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Mar;70(3):1775–1783. doi: 10.1128/jvi.70.3.1775-1783.1996

Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA.

G M Gilmartin 1, S L Hung 1, J D DeZazzo 1, E S Fleming 1, M J Imperiale 1
PMCID: PMC190003  PMID: 8627700

Abstract

The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter-proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini-MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors.

Full Text

The Full Text of this article is available as a PDF (369.1 KB).

Selected References

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

  1. Akusjärvi G., Persson H. Controls of RNA splicing and termination in the major late adenovirus transcription unit. Nature. 1981 Jul 30;292(5822):420–426. doi: 10.1038/292420a0. [DOI] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bienroth S., Wahle E., Suter-Crazzolara C., Keller W. Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors. J Biol Chem. 1991 Oct 15;266(29):19768–19776. [PubMed] [Google Scholar]
  4. Brown P. H., Tiley L. S., Cullen B. R. Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences. J Virol. 1991 Jun;65(6):3340–3343. doi: 10.1128/jvi.65.6.3340-3343.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carswell S., Alwine J. C. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol Cell Biol. 1989 Oct;9(10):4248–4258. doi: 10.1128/mcb.9.10.4248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chinnadurai G., Chinnadurai S., Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol. 1979 Nov;32(2):623–628. doi: 10.1128/jvi.32.2.623-628.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Christofori G., Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. doi: 10.1016/s0092-8674(88)91263-9. [DOI] [PubMed] [Google Scholar]
  9. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  10. DeZazzo J. D., Falck-Pedersen E., Imperiale M. J. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol Cell Biol. 1991 Dec;11(12):5977–5984. doi: 10.1128/mcb.11.12.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeZazzo J. D., Imperiale M. J. Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit. Mol Cell Biol. 1989 Nov;9(11):4951–4961. doi: 10.1128/mcb.9.11.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DeZazzo J. D., Kilpatrick J. E., Imperiale M. J. Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation. Mol Cell Biol. 1991 Mar;11(3):1624–1630. doi: 10.1128/mcb.11.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Falck-Pedersen E., Logan J. Regulation of poly(A) site selection in adenovirus. J Virol. 1989 Feb;63(2):532–541. doi: 10.1128/jvi.63.2.532-541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilmartin G. M., Fleming E. S., Oetjen J. Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR. EMBO J. 1992 Dec;11(12):4419–4428. doi: 10.1002/j.1460-2075.1992.tb05542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilmartin G. M., Fleming E. S., Oetjen J., Graveley B. R. CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition. Genes Dev. 1995 Jan 1;9(1):72–83. doi: 10.1101/gad.9.1.72. [DOI] [PubMed] [Google Scholar]
  16. Gilmartin G. M., McDevitt M. A., Nevins J. R. Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev. 1988 May;2(5):578–587. doi: 10.1101/gad.2.5.578. [DOI] [PubMed] [Google Scholar]
  17. Gilmartin G. M., Nevins J. R. An ordered pathway of assembly of components required for polyadenylation site recognition and processing. Genes Dev. 1989 Dec;3(12B):2180–2190. doi: 10.1101/gad.3.12b.2180. [DOI] [PubMed] [Google Scholar]
  18. Gilmartin G. M., Nevins J. R. Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol. 1991 May;11(5):2432–2438. doi: 10.1128/mcb.11.5.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hedley M. L., Maniatis T. Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro. Cell. 1991 May 17;65(4):579–586. doi: 10.1016/0092-8674(91)90090-l. [DOI] [PubMed] [Google Scholar]
  20. Keller W., Bienroth S., Lang K. M., Christofori G. Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA. EMBO J. 1991 Dec;10(13):4241–4249. doi: 10.1002/j.1460-2075.1991.tb05002.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin C. H., Patton J. G. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995 May;1(3):234–245. [PMC free article] [PubMed] [Google Scholar]
  22. Logan J., Shenk T. Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3655–3659. doi: 10.1073/pnas.81.12.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Manley J. L. Polyadenylation of mRNA precursors. Biochim Biophys Acta. 1988 May 6;950(1):1–12. doi: 10.1016/0167-4781(88)90067-x. [DOI] [PubMed] [Google Scholar]
  24. Mann K. P., Weiss E. A., Nevins J. R. Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor. Mol Cell Biol. 1993 Apr;13(4):2411–2419. doi: 10.1128/mcb.13.4.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  26. Moen P. T., Jr, Fox E., Bodnar J. W. Adenovirus and minute virus of mice DNAs are localized at the nuclear periphery. Nucleic Acids Res. 1990 Feb 11;18(3):513–520. doi: 10.1093/nar/18.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moyne G., Pichard E., Bernhard W. Localization of simian adenovirus 7 (SA 7) transcription and replication in lytic infection. An ultracytochemical and autoradiographical study. J Gen Virol. 1978 Jul;40(1):77–92. doi: 10.1099/0022-1317-40-1-77. [DOI] [PubMed] [Google Scholar]
  28. Murthy K. G., Manley J. L. Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem. 1992 Jul 25;267(21):14804–14811. [PubMed] [Google Scholar]
  29. Nevins J. R., Darnell J. E., Jr Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell. 1978 Dec;15(4):1477–1493. doi: 10.1016/0092-8674(78)90071-5. [DOI] [PubMed] [Google Scholar]
  30. Nevins J. R., Wilson M. C. Regulation of adenovirus-2 gene expression at the level of transcriptional termination and RNA processing. Nature. 1981 Mar 12;290(5802):113–118. doi: 10.1038/290113a0. [DOI] [PubMed] [Google Scholar]
  31. Peterson M. L. Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells. Mol Cell Biol. 1994 Dec;14(12):7891–7898. doi: 10.1128/mcb.14.12.7891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Prescott J. C., Falck-Pedersen E. Varied poly(A) site efficiency in the adenovirus major late transcription unit. J Biol Chem. 1992 Apr 25;267(12):8175–8181. [PubMed] [Google Scholar]
  33. Prescott J., Falck-Pedersen E. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol Cell Biol. 1994 Jul;14(7):4682–4693. doi: 10.1128/mcb.14.7.4682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Russnak R., Ganem D. Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. Genes Dev. 1990 May;4(5):764–776. doi: 10.1101/gad.4.5.764. [DOI] [PubMed] [Google Scholar]
  35. Schek N., Cooke C., Alwine J. C. Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses. Mol Cell Biol. 1992 Dec;12(12):5386–5393. doi: 10.1128/mcb.12.12.5386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shaw A. R., Ziff E. B. Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families. Cell. 1980 Dec;22(3):905–916. doi: 10.1016/0092-8674(80)90568-1. [DOI] [PubMed] [Google Scholar]
  37. Takagaki Y., Manley J. L., MacDonald C. C., Wilusz J., Shenk T. A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev. 1990 Dec;4(12A):2112–2120. doi: 10.1101/gad.4.12a.2112. [DOI] [PubMed] [Google Scholar]
  38. Takagaki Y., Ryner L. C., Manley J. L. Four factors are required for 3'-end cleavage of pre-mRNAs. Genes Dev. 1989 Nov;3(11):1711–1724. doi: 10.1101/gad.3.11.1711. [DOI] [PubMed] [Google Scholar]
  39. Takagaki Y., Ryner L. C., Manley J. L. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell. 1988 Mar 11;52(5):731–742. doi: 10.1016/0092-8674(88)90411-4. [DOI] [PubMed] [Google Scholar]
  40. Valcárcel J., Singh R., Zamore P. D., Green M. R. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature. 1993 Mar 11;362(6416):171–175. doi: 10.1038/362171a0. [DOI] [PubMed] [Google Scholar]
  41. Valsamakis A., Zeichner S., Carswell S., Alwine J. C. The human immunodeficiency virus type 1 polyadenylylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylylation. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2108–2112. doi: 10.1073/pnas.88.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wahle E. 3'-end cleavage and polyadenylation of mRNA precursors. Biochim Biophys Acta. 1995 Apr 4;1261(2):183–194. doi: 10.1016/0167-4781(94)00248-2. [DOI] [PubMed] [Google Scholar]
  43. Wahle E. A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation. Cell. 1991 Aug 23;66(4):759–768. doi: 10.1016/0092-8674(91)90119-j. [DOI] [PubMed] [Google Scholar]
  44. Wahle E., Keller W. The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. Annu Rev Biochem. 1992;61:419–440. doi: 10.1146/annurev.bi.61.070192.002223. [DOI] [PubMed] [Google Scholar]
  45. Weiss E. A., Gilmartin G. M., Nevins J. R. Poly(A) site efficiency reflects the stability of complex formation involving the downstream element. EMBO J. 1991 Jan;10(1):215–219. doi: 10.1002/j.1460-2075.1991.tb07938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wilson-Gunn S. I., Kilpatrick J. E., Imperiale M. J. Regulated adenovirus mRNA 3'-end formation in a coupled in vitro transcription-processing system. J Virol. 1992 Sep;66(9):5418–5424. doi: 10.1128/jvi.66.9.5418-5424.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilusz J., Shenk T., Takagaki Y., Manley J. L. A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates. Mol Cell Biol. 1990 Mar;10(3):1244–1248. doi: 10.1128/mcb.10.3.1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Younghusband H. B., Maundrell K. Adenovirus DNA is associated with the nuclear matrix of infected cells. J Virol. 1982 Aug;43(2):705–713. doi: 10.1128/jvi.43.2.705-713.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES