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. 1993 Feb;12(2):585–594. doi: 10.1002/j.1460-2075.1993.tb05690.x

Assembly of a processive messenger RNA polyadenylation complex.

S Bienroth 1, W Keller 1, E Wahle 1
PMCID: PMC413241  PMID: 8440247

Abstract

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.

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Selected References

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  1. Bachvarova R. F. A maternal tail of poly(A): the long and the short of it. Cell. 1992 Jun 12;69(6):895–897. doi: 10.1016/0092-8674(92)90606-d. [DOI] [PubMed] [Google Scholar]
  2. Bardwell V. J., Wickens M., Bienroth S., Keller W., Sproat B. S., Lamond A. I. Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition. Cell. 1991 Apr 5;65(1):125–133. doi: 10.1016/0092-8674(91)90414-t. [DOI] [PubMed] [Google Scholar]
  3. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  6. Bunick D., Zandomeni R., Ackerman S., Weinmann R. Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts. Cell. 1982 Jul;29(3):877–886. doi: 10.1016/0092-8674(82)90449-4. [DOI] [PubMed] [Google Scholar]
  7. Burgers P. M., Kornberg A. The cycling of Escherichia coli DNA polymerase III holoenzyme in replication. J Biol Chem. 1983 Jun 25;258(12):7669–7675. [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. 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]
  10. Conaway R. C., Conaway J. W. ATP activates transcription initiation from promoters by RNA polymerase II in a reversible step prior to RNA synthesis. J Biol Chem. 1988 Feb 25;263(6):2962–2968. [PubMed] [Google Scholar]
  11. Dominski Z., Kole R. Cooperation of pre-mRNA sequence elements in splice site selection. Mol Cell Biol. 1992 May;12(5):2108–2114. doi: 10.1128/mcb.12.5.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fox C. A., Sheets M. D., Wickens M. P. Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU. Genes Dev. 1989 Dec;3(12B):2151–2162. doi: 10.1101/gad.3.12b.2151. [DOI] [PubMed] [Google Scholar]
  13. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  14. Frendewey D., Krämer A., Keller W. Different small nuclear ribonucleoprotein particles are involved in different steps of splicing complex formation. Cold Spring Harb Symp Quant Biol. 1987;52:287–298. doi: 10.1101/sqb.1987.052.01.034. [DOI] [PubMed] [Google Scholar]
  15. Gallie D. R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 1991 Nov;5(11):2108–2116. doi: 10.1101/gad.5.11.2108. [DOI] [PubMed] [Google Scholar]
  16. Ge H., Manley J. L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. doi: 10.1016/0092-8674(90)90236-8. [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. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Keller W. The RNA lariat: a new ring to the splicing of mRNA precursors. Cell. 1984 Dec;39(3 Pt 2):423–425. doi: 10.1016/0092-8674(84)90449-5. [DOI] [PubMed] [Google Scholar]
  25. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  26. Krainer A. R., Conway G. C., Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. doi: 10.1016/0092-8674(90)90237-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  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., 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]
  30. 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]
  31. O'Donnell M. Accessory protein function in the DNA polymerase III holoenzyme from E. coli. Bioessays. 1992 Feb;14(2):105–111. doi: 10.1002/bies.950140206. [DOI] [PubMed] [Google Scholar]
  32. Ohshima Y., Gotoh Y. Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences. J Mol Biol. 1987 May 20;195(2):247–259. doi: 10.1016/0022-2836(87)90647-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Proudfoot N. Poly(A) signals. Cell. 1991 Feb 22;64(4):671–674. doi: 10.1016/0092-8674(91)90495-k. [DOI] [PubMed] [Google Scholar]
  35. Raabe T., Bollum F. J., Manley J. L. Primary structure and expression of bovine poly(A) polymerase. Nature. 1991 Sep 19;353(6341):229–234. doi: 10.1038/353229a0. [DOI] [PubMed] [Google Scholar]
  36. Rowen L., Kornberg A. A ribo-deoxyribonucleotide primer synthesized by primase. J Biol Chem. 1978 Feb 10;253(3):770–774. [PubMed] [Google Scholar]
  37. 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]
  38. Ryner L. C., Manley J. L. Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro. Mol Cell Biol. 1987 Jan;7(1):495–503. doi: 10.1128/mcb.7.1.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  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. 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]
  44. Stukenberg P. T., Studwell-Vaughan P. S., O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991 Jun 15;266(17):11328–11334. [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Tsiapalis C. M., Dorson J. W., Bollum F. J. Purification of terminal riboadenylate transferase from calf thymus gland. J Biol Chem. 1975 Jun 25;250(12):4486–4496. [PubMed] [Google Scholar]
  49. 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]
  50. Wahle E., Martin G., Schiltz E., Keller W. Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. EMBO J. 1991 Dec;10(13):4251–4257. doi: 10.1002/j.1460-2075.1991.tb05003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wahle E. Purification and characterization of a mammalian polyadenylate polymerase involved in the 3' end processing of messenger RNA precursors. J Biol Chem. 1991 Feb 15;266(5):3131–3139. [PubMed] [Google Scholar]
  52. Wahle E. The end of the message: 3'-end processing leading to polyadenylated messenger RNA. Bioessays. 1992 Feb;14(2):113–118. doi: 10.1002/bies.950140208. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Wickens M. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biochem Sci. 1990 Aug;15(8):320–324. doi: 10.1016/0968-0004(90)90022-4. [DOI] [PubMed] [Google Scholar]
  56. Wickner W., Kornberg A. DNA polymerase 3 star requires ATP to start synthesis on a primed DNA. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3679–3683. doi: 10.1073/pnas.70.12.3679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Wu C. A., Zechner E. L., Marians K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. J Biol Chem. 1992 Feb 25;267(6):4030–4044. [PubMed] [Google Scholar]
  59. 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]
  60. 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]

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