Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Sep;12(9):3699–3705. doi: 10.1128/mcb.12.9.3699

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

A Valsamakis 1, N Schek 1, J C Alwine 1
PMCID: PMC360226  PMID: 1508176

Abstract

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.

Full text

PDF
3702

Images in this article

3701Image
on p.3701
3702Image
on p.3702
3702Image
on p.3702
3703Image
on p.3703

Selected References

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

  1. Ahmed Y. F., Gilmartin G. M., Hanly S. M., Nevins J. R., Greene W. C. The HTLV-I Rex response element mediates a novel form of mRNA polyadenylation. Cell. 1991 Feb 22;64(4):727–737. doi: 10.1016/0092-8674(91)90502-p. [DOI] [PubMed] [Google Scholar]
  2. Bar-Shira A., Panet A., Honigman A. An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing. J Virol. 1991 Oct;65(10):5165–5173. doi: 10.1128/jvi.65.10.5165-5173.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhat B. M., Wold W. S. ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus. Mol Cell Biol. 1985 Nov;5(11):3183–3193. doi: 10.1128/mcb.5.11.3183. [DOI] [PMC free article] [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. Böhnlein S., Hauber J., Cullen B. R. Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat. J Virol. 1989 Jan;63(1):421–424. doi: 10.1128/jvi.63.1.421-424.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Cole C. N., Stacy T. P. Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation. Mol Cell Biol. 1985 Aug;5(8):2104–2113. doi: 10.1128/mcb.5.8.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conway L., Wickens M. A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3949–3953. doi: 10.1073/pnas.82.12.3949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Falck-Pedersen E., Logan J., Shenk T., Darnell J. E., Jr Transcription termination within the E1A gene of adenovirus induced by insertion of the mouse beta-major globin terminator element. Cell. 1985 Apr;40(4):897–905. doi: 10.1016/0092-8674(85)90349-6. [DOI] [PubMed] [Google Scholar]
  13. Fitzgerald M., Shenk T. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell. 1981 Apr;24(1):251–260. doi: 10.1016/0092-8674(81)90521-3. [DOI] [PubMed] [Google Scholar]
  14. Galli G., Guise J. W., McDevitt M. A., Tucker P. W., Nevins J. R. Relative position and strengths of poly(A) sites as well as transcription termination are critical to membrane versus secreted mu-chain expression during B-cell development. Genes Dev. 1987 Jul;1(5):471–481. doi: 10.1101/gad.1.5.471. [DOI] [PubMed] [Google Scholar]
  15. Galli G., Guise J., Tucker P. W., Nevins J. R. Poly(A) site choice rather than splice site choice governs the regulated production of IgM heavy-chain RNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2439–2443. doi: 10.1073/pnas.85.8.2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gil A., Proudfoot N. J. A sequence downstream of AAUAAA is required for rabbit beta-globin mRNA 3'-end formation. 1984 Nov 29-Dec 5Nature. 312(5993):473–474. doi: 10.1038/312473a0. [DOI] [PubMed] [Google Scholar]
  17. Gil A., Proudfoot N. J. Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987 May 8;49(3):399–406. doi: 10.1016/0092-8674(87)90292-3. [DOI] [PubMed] [Google Scholar]
  18. Hart R. P., McDevitt M. A., Ali H., Nevins J. R. Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites. Mol Cell Biol. 1985 Nov;5(11):2975–2983. doi: 10.1128/mcb.5.11.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hart R. P., McDevitt M. A., Nevins J. R. Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences. Cell. 1985 Dec;43(3 Pt 2):677–683. doi: 10.1016/0092-8674(85)90240-5. [DOI] [PubMed] [Google Scholar]
  20. Heath C. V., Denome R. M., Cole C. N. Spatial constraints on polyadenylation signal function. J Biol Chem. 1990 Jun 5;265(16):9098–9104. [PubMed] [Google Scholar]
  21. Iwasaki K., Temin H. M. The efficiency of RNA 3'-end formation is determined by the distance between the cap site and the poly(A) site in spleen necrosis virus. Genes Dev. 1990 Dec;4(12B):2299–2307. doi: 10.1101/gad.4.12b.2299. [DOI] [PubMed] [Google Scholar]
  22. McDevitt M. A., Hart R. P., Wong W. W., Nevins J. R. Sequences capable of restoring poly(A) site function define two distinct downstream elements. EMBO J. 1986 Nov;5(11):2907–2913. doi: 10.1002/j.1460-2075.1986.tb04586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McDevitt M. A., Imperiale M. J., Ali H., Nevins J. R. Requirement of a downstream sequence for generation of a poly(A) addition site. Cell. 1984 Jul;37(3):993–999. doi: 10.1016/0092-8674(84)90433-1. [DOI] [PubMed] [Google Scholar]
  24. McLauchlan J., Gaffney D., Whitton J. L., Clements J. B. The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. Nucleic Acids Res. 1985 Feb 25;13(4):1347–1368. doi: 10.1093/nar/13.4.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McLauchlan J., Simpson S., Clements J. B. Herpes simplex virus induces a processing factor that stimulates poly(A) site usage. Cell. 1989 Dec 22;59(6):1093–1105. doi: 10.1016/0092-8674(89)90765-4. [DOI] [PubMed] [Google Scholar]
  26. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Inhibition of RNA cleavage but not polyadenylation by a point mutation in mRNA 3' consensus sequence AAUAAA. Nature. 1983 Oct 13;305(5935):600–605. doi: 10.1038/305600a0. [DOI] [PubMed] [Google Scholar]
  27. Moore C. L. Preparation of mammalian extracts active in polyadenylation. Methods Enzymol. 1990;181:49–74. doi: 10.1016/0076-6879(90)81112-8. [DOI] [PubMed] [Google Scholar]
  28. Moore C. L., Sharp P. A. Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell. 1985 Jul;41(3):845–855. doi: 10.1016/s0092-8674(85)80065-9. [DOI] [PubMed] [Google Scholar]
  29. Moore C. L., Sharp P. A. Site-specific polyadenylation in a cell-free reaction. Cell. 1984 Mar;36(3):581–591. doi: 10.1016/0092-8674(84)90337-4. [DOI] [PubMed] [Google Scholar]
  30. Peterson M. L., Perry R. P. The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice. Mol Cell Biol. 1989 Feb;9(2):726–738. doi: 10.1128/mcb.9.2.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Ryner L. C., Takagaki Y., Manley J. L. Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly. Mol Cell Biol. 1989 Apr;9(4):1759–1771. doi: 10.1128/mcb.9.4.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sadofsky M., Alwine J. C. Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site. Mol Cell Biol. 1984 Aug;4(8):1460–1468. doi: 10.1128/mcb.4.8.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sadofsky M., Connelly S., Manley J. L., Alwine J. C. Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing. Mol Cell Biol. 1985 Oct;5(10):2713–2719. doi: 10.1128/mcb.5.10.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sanfaçon H., Brodmann P., Hohn T. A dissection of the cauliflower mosaic virus polyadenylation signal. Genes Dev. 1991 Jan;5(1):141–149. doi: 10.1101/gad.5.1.141. [DOI] [PubMed] [Google Scholar]
  37. Sperry A. O., Berget S. M. In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence. Mol Cell Biol. 1986 Dec;6(12):4734–4741. doi: 10.1128/mcb.6.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Weichs an der Glon C., Monks J., Proudfoot N. J. Occlusion of the HIV poly(A) site. Genes Dev. 1991 Feb;5(2):244–253. doi: 10.1101/gad.5.2.244. [DOI] [PubMed] [Google Scholar]
  40. Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci. 1990 Jul;15(7):277–281. doi: 10.1016/0968-0004(90)90054-f. [DOI] [PubMed] [Google Scholar]
  41. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]
  42. Wilusz J., Shenk T. A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal. Mol Cell Biol. 1990 Dec;10(12):6397–6407. doi: 10.1128/mcb.10.12.6397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zarkower D., Stephenson P., Sheets M., Wickens M. The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro. Mol Cell Biol. 1986 Jul;6(7):2317–2323. doi: 10.1128/mcb.6.7.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zeichner S. L., Kim J. Y., Alwine J. C. Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1991 May;65(5):2436–2444. doi: 10.1128/jvi.65.5.2436-2444.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang F., Cole C. N. Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA. Mol Cell Biol. 1987 Sep;7(9):3277–3286. doi: 10.1128/mcb.7.9.3277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang F., Denome R. M., Cole C. N. Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations. Mol Cell Biol. 1986 Dec;6(12):4611–4623. doi: 10.1128/mcb.6.12.4611. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES