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. 2000 May;6(5):768–777. doi: 10.1017/s135583820099246x

Isolation and characterization of polyadenylation complexes assembled in vitro.

K L Veraldi 1, G Edwalds-Gilbert 1, C C MacDonald 1, A M Wallace 1, C Milcarek 1
PMCID: PMC1369956  PMID: 10836797

Abstract

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.

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Selected References

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  1. Bennett M., Piñol-Roma S., Staknis D., Dreyfuss G., Reed R. Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro. Mol Cell Biol. 1992 Jul;12(7):3165–3175. doi: 10.1128/mcb.12.7.3165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beyer K., Dandekar T., Keller W. RNA ligands selected by cleavage stimulation factor contain distinct sequence motifs that function as downstream elements in 3'-end processing of pre-mRNA. J Biol Chem. 1997 Oct 17;272(42):26769–26779. doi: 10.1074/jbc.272.42.26769. [DOI] [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. Chen F., MacDonald C. C., Wilusz J. Cleavage site determinants in the mammalian polyadenylation signal. Nucleic Acids Res. 1995 Jul 25;23(14):2614–2620. doi: 10.1093/nar/23.14.2614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Dantonel J. C., Murthy K. G., Manley J. L., Tora L. Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA. Nature. 1997 Sep 25;389(6649):399–402. doi: 10.1038/38763. [DOI] [PubMed] [Google Scholar]
  7. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edwalds-Gilbert G., Milcarek C. Regulation of poly(A) site use during mouse B-cell development involves a change in the binding of a general polyadenylation factor in a B-cell stage-specific manner. Mol Cell Biol. 1995 Nov;15(11):6420–6429. doi: 10.1128/mcb.15.11.6420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edwalds-Gilbert G., Veraldi K. L., Milcarek C. Alternative poly(A) site selection in complex transcription units: means to an end? Nucleic Acids Res. 1997 Jul 1;25(13):2547–2561. doi: 10.1093/nar/25.13.2547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Grabowski P. J., Sharp P. A. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 1986 Sep 19;233(4770):1294–1299. doi: 10.1126/science.3638792. [DOI] [PubMed] [Google Scholar]
  12. Gunderson S. I., Polycarpou-Schwarz M., Mattaj I. W. U1 snRNP inhibits pre-mRNA polyadenylation through a direct interaction between U1 70K and poly(A) polymerase. Mol Cell. 1998 Jan;1(2):255–264. doi: 10.1016/s1097-2765(00)80026-x. [DOI] [PubMed] [Google Scholar]
  13. Gunderson S. I., Vagner S., Polycarpou-Schwarz M., Mattaj I. W. Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation. Genes Dev. 1997 Mar 15;11(6):761–773. doi: 10.1101/gad.11.6.761. [DOI] [PubMed] [Google Scholar]
  14. Jenny A., Hauri H. P., Keller W. Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit. Mol Cell Biol. 1994 Dec;14(12):8183–8190. doi: 10.1128/mcb.14.12.8183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Keller W. No end yet to messenger RNA 3' processing! Cell. 1995 Jun 16;81(6):829–832. doi: 10.1016/0092-8674(95)90001-2. [DOI] [PubMed] [Google Scholar]
  17. Lutz C. S., Murthy K. G., Schek N., O'Connor J. P., Manley J. L., Alwine J. C. Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro. Genes Dev. 1996 Feb 1;10(3):325–337. doi: 10.1101/gad.10.3.325. [DOI] [PubMed] [Google Scholar]
  18. MacDonald C. C., Wilusz J., Shenk T. The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location. Mol Cell Biol. 1994 Oct;14(10):6647–6654. doi: 10.1128/mcb.14.10.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martincic K., Campbell R., Edwalds-Gilbert G., Souan L., Lotze M. T., Milcarek C. Increase in the 64-kDa subunit of the polyadenylation/cleavage stimulatory factor during the G0 to S phase transition. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11095–11100. doi: 10.1073/pnas.95.19.11095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matis S. A., Martincic K., Milcarek C. B-lineage regulated polyadenylation occurs on weak poly(A) sites regardless of sequence composition at the cleavage and downstream regions. Nucleic Acids Res. 1996 Dec 1;24(23):4684–4692. doi: 10.1093/nar/24.23.4684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matthews H. R. The application of Cerenkov counting to column chromatography of 32P labelled substances. J Chromatogr. 1968 Aug 27;36(3):302–308. doi: 10.1016/s0021-9673(01)92946-2. [DOI] [PubMed] [Google Scholar]
  22. McCracken S., Fong N., Yankulov K., Ballantyne S., Pan G., Greenblatt J., Patterson S. D., Wickens M., Bentley D. L. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature. 1997 Jan 23;385(6614):357–361. doi: 10.1038/385357a0. [DOI] [PubMed] [Google Scholar]
  23. Milcarek C., Suda-Hartman M., Croll S. C. Changes in abundance of IgG 2a mRNA in the nucleus and cytoplasm of a murine B-lymphoma before and after fusion to a myeloma cell. Mol Immunol. 1996 May-Jun;33(7-8):691–701. doi: 10.1016/0161-5890(96)00009-0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Murthy K. G., Manley J. L. The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. Genes Dev. 1995 Nov 1;9(21):2672–2683. doi: 10.1101/gad.9.21.2672. [DOI] [PubMed] [Google Scholar]
  28. Reed R. Protein composition of mammalian spliceosomes assembled in vitro. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8031–8035. doi: 10.1073/pnas.87.20.8031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rüegsegger U., Beyer K., Keller W. Purification and characterization of human cleavage factor Im involved in the 3' end processing of messenger RNA precursors. J Biol Chem. 1996 Mar 15;271(11):6107–6113. doi: 10.1074/jbc.271.11.6107. [DOI] [PubMed] [Google Scholar]
  30. Rüegsegger U., Blank D., Keller W. Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. Mol Cell. 1998 Jan;1(2):243–253. doi: 10.1016/s1097-2765(00)80025-8. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Takagaki Y., MacDonald C. C., Shenk T., Manley J. L. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1403–1407. doi: 10.1073/pnas.89.4.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Takagaki Y., Manley J. L. Complex protein interactions within the human polyadenylation machinery identify a novel component. Mol Cell Biol. 2000 Mar;20(5):1515–1525. doi: 10.1128/mcb.20.5.1515-1525.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takagaki Y., Manley J. L. RNA recognition by the human polyadenylation factor CstF. Mol Cell Biol. 1997 Jul;17(7):3907–3914. doi: 10.1128/mcb.17.7.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Wallace A. M., Dass B., Ravnik S. E., Tonk V., Jenkins N. A., Gilbert D. J., Copeland N. G., MacDonald C. C. Two distinct forms of the 64,000 Mr protein of the cleavage stimulation factor are expressed in mouse male germ cells. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6763–6768. doi: 10.1073/pnas.96.12.6763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Zhao J., Hyman L., Moore C. Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev. 1999 Jun;63(2):405–445. doi: 10.1128/mmbr.63.2.405-445.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

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