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. 1990 Jan;10(1):295–302. doi: 10.1128/mcb.10.1.295

Polyadenylation-specific complexes undergo a transition early in the polymerization of a poly(A) tail.

V J Bardwell 1, M Wickens 1
PMCID: PMC360737  PMID: 2294406

Abstract

We have analyzed several properties of the complex that forms between RNAs that end at the poly(A) site of simian virus 40 late mRNA and factors present in a HeLa cell nuclear extract. Formation of this polyadenylation-specific complex requires the sequence AAUAAA and a proximal 3' end. We have observed three changes in the polyadenylation complex early in the addition of the poly(A) tail. First, the complex becomes heparin sensitive after the addition of approximately 10 adenosines. Second, a 68-kilodalton protein present in the complex, which can be cross-linked by UV light to the RNA before polyadenylation has begun, no longer can be cross-linked after approximately 10 adenosines have been added. Third, after 30 adenosines have been added, the AAUAAA sequence becomes accessible to a complementary oligonucleotide and RNase H. This accessibility gradually increases with longer poly(A) tail lengths until, with the addition of 60 A's, all substrates are accessible at AAUAAA. Sheets and Wickens (Genes Dev. 3:1401-1412, 1989) have recently demonstrated two phases in the addition of a poly(A) tail: the first requires AAUAAA, whereas the second is independent of AAUAAA but requires a short oligo(A) primer. The data reported here further support a biphasic model for poly(A) addition and may indicate disengagement of specific factors from AAUAAA after the initiation phase.

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Selected References

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

  1. 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]
  2. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Christofori G., Keller W. Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. Mol Cell Biol. 1989 Jan;9(1):193–203. doi: 10.1128/mcb.9.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Hashimoto C., Steitz J. A. A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro. Cell. 1986 May 23;45(4):581–591. doi: 10.1016/0092-8674(86)90290-4. [DOI] [PubMed] [Google Scholar]
  7. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  8. Humphrey T., Christofori G., Lucijanic V., Keller W. Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes. EMBO J. 1987 Dec 20;6(13):4159–4168. doi: 10.1002/j.1460-2075.1987.tb02762.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. McDevitt M. A., Gilmartin G. M., Reeves W. H., Nevins J. R. Multiple factors are required for poly(A) addition to a mRNA 3' end. Genes Dev. 1988 May;2(5):588–597. doi: 10.1101/gad.2.5.588. [DOI] [PubMed] [Google Scholar]
  11. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moore C. L., Chen J., Whoriskey J. Two proteins crosslinked to RNA containing the adenovirus L3 poly(A) site require the AAUAAA sequence for binding. EMBO J. 1988 Oct;7(10):3159–3169. doi: 10.1002/j.1460-2075.1988.tb03183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Moore C. L., Skolnik-David H., Sharp P. A. Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site. EMBO J. 1986 Aug;5(8):1929–1938. doi: 10.1002/j.1460-2075.1986.tb04446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moore C. L., Skolnik-David H., Sharp P. A. Sedimentation analysis of polyadenylation-specific complexes. Mol Cell Biol. 1988 Jan;8(1):226–233. doi: 10.1128/mcb.8.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pikielny C. W., Rosbash M. Specific small nuclear RNAs are associated with yeast spliceosomes. Cell. 1986 Jun 20;45(6):869–877. doi: 10.1016/0092-8674(86)90561-1. [DOI] [PubMed] [Google Scholar]
  17. Ruskin B., Green M. R. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell. 1985 Nov;43(1):131–142. doi: 10.1016/0092-8674(85)90018-2. [DOI] [PubMed] [Google Scholar]
  18. Ryner L. C., Takagaki Y., Manley J. L. Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation. Mol Cell Biol. 1989 Oct;9(10):4229–4238. doi: 10.1128/mcb.9.10.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sheets M. D., Wickens M. Two phases in the addition of a poly(A) tail. Genes Dev. 1989 Sep;3(9):1401–1412. doi: 10.1101/gad.3.9.1401. [DOI] [PubMed] [Google Scholar]
  20. Skolnik-David H., Moore C. L., Sharp P. A. Electrophoretic separation of polyadenylation-specific complexes. Genes Dev. 1987 Sep;1(7):672–682. doi: 10.1101/gad.1.7.672. [DOI] [PubMed] [Google Scholar]
  21. Stefano J. E., Adams D. E. Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro. Mol Cell Biol. 1988 May;8(5):2052–2062. doi: 10.1128/mcb.8.5.2052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Wilusz J., Shenk T. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell. 1988 Jan 29;52(2):221–228. doi: 10.1016/0092-8674(88)90510-7. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Zarkower D., Wickens M. Formation of mRNA 3' termini: stability and dissociation of a complex involving the AAUAAA sequence. EMBO J. 1987 Jan;6(1):177–186. doi: 10.1002/j.1460-2075.1987.tb04736.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zarkower D., Wickens M. Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3' termini in vitro. EMBO J. 1987 Dec 20;6(13):4185–4192. doi: 10.1002/j.1460-2075.1987.tb02765.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]

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