Skip to main content
NCBI home page
RNA logoLink to RNA
. 2002 Sep;8(9):1102–1111. doi: 10.1017/s1355838202025037

Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD.

Andrew Emili 1, Michael Shales 1, Susan McCracken 1, Weijun Xie 1, Philip W Tucker 1, Ryuji Kobayashi 1, Benjamin J Blencowe 1, C James Ingles 1
PMCID: PMC1370324  PMID: 12358429

Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

Full Text

The Full Text of this article is available as a PDF (223.0 KB).

Selected References

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

  1. Allison L. A., Ingles C. J. Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2794–2798. doi: 10.1073/pnas.86.8.2794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison L. A., Moyle M., Shales M., Ingles C. J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985 Sep;42(2):599–610. doi: 10.1016/0092-8674(85)90117-5. [DOI] [PubMed] [Google Scholar]
  3. Barillà D., Lee B. A., Proudfoot N. J. Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2001 Jan 9;98(2):445–450. doi: 10.1073/pnas.98.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Basu A., Dong B., Krainer A. R., Howe C. C. The intracisternal A-particle proximal enhancer-binding protein activates transcription and is identical to the RNA- and DNA-binding protein p54nrb/NonO. Mol Cell Biol. 1997 Feb;17(2):677–686. doi: 10.1128/mcb.17.2.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blencowe B. J., Issner R., Nickerson J. A., Sharp P. A. A coactivator of pre-mRNA splicing. Genes Dev. 1998 Apr 1;12(7):996–1009. doi: 10.1101/gad.12.7.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conaway J. W., Shilatifard A., Dvir A., Conaway R. C. Control of elongation by RNA polymerase II. Trends Biochem Sci. 2000 Aug;25(8):375–380. doi: 10.1016/s0968-0004(00)01615-7. [DOI] [PubMed] [Google Scholar]
  7. Corden J. L., Cadena D. L., Ahearn J. M., Jr, Dahmus M. E. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7934–7938. doi: 10.1073/pnas.82.23.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cramer P., Cáceres J. F., Cazalla D., Kadener S., Muro A. F., Baralle F. E., Kornblihtt A. R. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol Cell. 1999 Aug;4(2):251–258. doi: 10.1016/s1097-2765(00)80372-x. [DOI] [PubMed] [Google Scholar]
  9. Cramer P., Srebrow A., Kadener S., Werbajh S., de la Mata M., Melen G., Nogués G., Kornblihtt A. R. Coordination between transcription and pre-mRNA processing. FEBS Lett. 2001 Jun 8;498(2-3):179–182. doi: 10.1016/s0014-5793(01)02485-1. [DOI] [PubMed] [Google Scholar]
  10. Dahmus M. E. Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem. 1996 Aug 9;271(32):19009–19012. doi: 10.1074/jbc.271.32.19009. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Dong B., Horowitz D. S., Kobayashi R., Krainer A. R. Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6. Nucleic Acids Res. 1993 Aug 25;21(17):4085–4092. doi: 10.1093/nar/21.17.4085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dye B. T., Patton J. G. An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles. Exp Cell Res. 2001 Feb 1;263(1):131–144. doi: 10.1006/excr.2000.5097. [DOI] [PubMed] [Google Scholar]
  14. Emili A., Ingles C. J. Promoter-dependent photocross-linking of the acidic transcriptional activator E2F-1 to the TATA-binding protein. J Biol Chem. 1995 Jun 9;270(23):13674–13680. doi: 10.1074/jbc.270.23.13674. [DOI] [PubMed] [Google Scholar]
  15. Fong Y. W., Zhou Q. Stimulatory effect of splicing factors on transcriptional elongation. Nature. 2001 Dec 20;414(6866):929–933. doi: 10.1038/414929a. [DOI] [PubMed] [Google Scholar]
  16. Fox Archa H., Lam Yun Wah, Leung Anthony K. L., Lyon Carol E., Andersen Jens, Mann Matthias, Lamond Angus I. Paraspeckles: a novel nuclear domain. Curr Biol. 2002 Jan 8;12(1):13–25. doi: 10.1016/s0960-9822(01)00632-7. [DOI] [PubMed] [Google Scholar]
  17. Gerber H. P., Hagmann M., Seipel K., Georgiev O., West M. A., Litingtung Y., Schaffner W., Corden J. L. RNA polymerase II C-terminal domain required for enhancer-driven transcription. Nature. 1995 Apr 13;374(6523):660–662. doi: 10.1038/374660a0. [DOI] [PubMed] [Google Scholar]
  18. Gozani O., Patton J. G., Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. doi: 10.1002/j.1460-2075.1994.tb06638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirose Y., Manley J. L. RNA polymerase II and the integration of nuclear events. Genes Dev. 2000 Jun 15;14(12):1415–1429. [PubMed] [Google Scholar]
  20. Hirose Y., Manley J. L. RNA polymerase II is an essential mRNA polyadenylation factor. Nature. 1998 Sep 3;395(6697):93–96. doi: 10.1038/25786. [DOI] [PubMed] [Google Scholar]
  21. Hirose Y., Tacke R., Manley J. L. Phosphorylated RNA polymerase II stimulates pre-mRNA splicing. Genes Dev. 1999 May 15;13(10):1234–1239. doi: 10.1101/gad.13.10.1234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ho C. K., Sriskanda V., McCracken S., Bentley D., Schwer B., Shuman S. The guanylyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II. J Biol Chem. 1998 Apr 17;273(16):9577–9585. doi: 10.1074/jbc.273.16.9577. [DOI] [PubMed] [Google Scholar]
  23. Kim E., Du L., Bregman D. B., Warren S. L. Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J Cell Biol. 1997 Jan 13;136(1):19–28. doi: 10.1083/jcb.136.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  25. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  26. Komarnitsky P., Cho E. J., Buratowski S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 2000 Oct 1;14(19):2452–2460. doi: 10.1101/gad.824700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lu H., Flores O., Weinmann R., Reinberg D. The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10004–10008. doi: 10.1073/pnas.88.22.10004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lutz C. S., Cooke C., O'Connor J. P., Kobayashi R., Alwine J. C. The snRNP-free U1A (SF-A) complex(es): identification of the largest subunit as PSF, the polypyrimidine-tract binding protein-associated splicing factor. RNA. 1998 Dec;4(12):1493–1499. doi: 10.1017/s1355838298981183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mathur M., Tucker P. W., Samuels H. H. PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors. Mol Cell Biol. 2001 Apr;21(7):2298–2311. doi: 10.1128/MCB.21.7.2298-2311.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McCracken S., Fong N., Rosonina E., Yankulov K., Brothers G., Siderovski D., Hessel A., Foster S., Shuman S., Bentley D. L. 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 1997 Dec 15;11(24):3306–3318. doi: 10.1101/gad.11.24.3306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. McCracken Susan, Lambermon Mark, Blencowe Benjamin J. SRm160 splicing coactivator promotes transcript 3'-end cleavage. Mol Cell Biol. 2002 Jan;22(1):148–160. doi: 10.1128/MCB.22.1.148-160.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Misteli T., Spector D. L. RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. Mol Cell. 1999 Jun;3(6):697–705. doi: 10.1016/s1097-2765(01)80002-2. [DOI] [PubMed] [Google Scholar]
  34. Morris D. P., Greenleaf A. L. The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II. J Biol Chem. 2000 Dec 22;275(51):39935–39943. doi: 10.1074/jbc.M004118200. [DOI] [PubMed] [Google Scholar]
  35. Mortillaro M. J., Blencowe B. J., Wei X., Nakayasu H., Du L., Warren S. L., Sharp P. A., Berezney R. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8253–8257. doi: 10.1073/pnas.93.16.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moyle M., Lee J. S., Anderson W. F., Ingles C. J. The C-terminal domain of the largest subunit of RNA polymerase II and transcription initiation. Mol Cell Biol. 1989 Dec;9(12):5750–5753. doi: 10.1128/mcb.9.12.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nonet M., Sweetser D., Young R. A. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987 Sep 11;50(6):909–915. doi: 10.1016/0092-8674(87)90517-4. [DOI] [PubMed] [Google Scholar]
  38. Ossipow V., Tassan J. P., Nigg E. A., Schibler U. A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell. 1995 Oct 6;83(1):137–146. doi: 10.1016/0092-8674(95)90242-2. [DOI] [PubMed] [Google Scholar]
  39. Otto H., Dreger M., Bengtsson L., Hucho F. Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope. Eur J Biochem. 2001 Jan;268(2):420–428. doi: 10.1046/j.1432-1033.2001.01901.x. [DOI] [PubMed] [Google Scholar]
  40. Pan G., Aso T., Greenblatt J. Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. J Biol Chem. 1997 Sep 26;272(39):24563–24571. doi: 10.1074/jbc.272.39.24563. [DOI] [PubMed] [Google Scholar]
  41. Patton J. G., Porro E. B., Galceran J., Tempst P., Nadal-Ginard B. Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev. 1993 Mar;7(3):393–406. doi: 10.1101/gad.7.3.393. [DOI] [PubMed] [Google Scholar]
  42. Patturajan M., Schulte R. J., Sefton B. M., Berezney R., Vincent M., Bensaude O., Warren S. L., Corden J. L. Growth-related changes in phosphorylation of yeast RNA polymerase II. J Biol Chem. 1998 Feb 20;273(8):4689–4694. doi: 10.1074/jbc.273.8.4689. [DOI] [PubMed] [Google Scholar]
  43. Patturajan M., Wei X., Berezney R., Corden J. L. A nuclear matrix protein interacts with the phosphorylated C-terminal domain of RNA polymerase II. Mol Cell Biol. 1998 Apr;18(4):2406–2415. doi: 10.1128/mcb.18.4.2406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pei Y., Hausmann S., Ho C. K., Schwer B., Shuman S. The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes. J Biol Chem. 2001 May 31;276(30):28075–28082. doi: 10.1074/jbc.M102170200. [DOI] [PubMed] [Google Scholar]
  45. Peterson S. R., Dvir A., Anderson C. W., Dynan W. S. DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats. Genes Dev. 1992 Mar;6(3):426–438. doi: 10.1101/gad.6.3.426. [DOI] [PubMed] [Google Scholar]
  46. Rodrigues J. P., Rode M., Gatfield D., Blencowe B. J., Carmo-Fonseca M., Izaurralde E. REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc Natl Acad Sci U S A. 2001 Jan 23;98(3):1030–1035. doi: 10.1073/pnas.031586198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scafe C., Chao D., Lopes J., Hirsch J. P., Henry S., Young R. A. RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals. Nature. 1990 Oct 4;347(6292):491–494. doi: 10.1038/347491a0. [DOI] [PubMed] [Google Scholar]
  48. Schroeder S. C., Schwer B., Shuman S., Bentley D. Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Dev. 2000 Oct 1;14(19):2435–2440. doi: 10.1101/gad.836300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sopta M., Carthew R. W., Greenblatt J. Isolation of three proteins that bind to mammalian RNA polymerase II. J Biol Chem. 1985 Aug 25;260(18):10353–10360. [PubMed] [Google Scholar]
  50. Steinmetz E. J. Pre-mRNA processing and the CTD of RNA polymerase II: the tail that wags the dog? Cell. 1997 May 16;89(4):491–494. doi: 10.1016/s0092-8674(00)80230-5. [DOI] [PubMed] [Google Scholar]
  51. Straub T., Grue P., Uhse A., Lisby M., Knudsen B. R., Tange T. O., Westergaard O., Boege F. The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction. J Biol Chem. 1998 Oct 9;273(41):26261–26264. doi: 10.1074/jbc.273.41.26261. [DOI] [PubMed] [Google Scholar]
  52. Thompson N. E., Aronson D. B., Burgess R. R. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody. J Biol Chem. 1990 Apr 25;265(12):7069–7077. [PubMed] [Google Scholar]
  53. Urban R. J., Bodenburg Y., Kurosky A., Wood T. G., Gasic S. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element. Mol Endocrinol. 2000 Jun;14(6):774–782. doi: 10.1210/mend.14.6.0485. [DOI] [PubMed] [Google Scholar]
  54. Vincent M., Lauriault P., Dubois M. F., Lavoie S., Bensaude O., Chabot B. The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes. Nucleic Acids Res. 1996 Dec 1;24(23):4649–4652. doi: 10.1093/nar/24.23.4649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Virbasius C. M., Wagner S., Green M. R. A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins. Mol Cell. 1999 Aug;4(2):219–228. doi: 10.1016/s1097-2765(00)80369-x. [DOI] [PubMed] [Google Scholar]
  56. Wagner E. J., Garcia-Blanco M. A. Polypyrimidine tract binding protein antagonizes exon definition. Mol Cell Biol. 2001 May;21(10):3281–3288. doi: 10.1128/MCB.21.10.3281-3288.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang R., Kobayashi R., Bishop J. M. Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8425–8430. doi: 10.1073/pnas.93.16.8425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yang Y. S., Hanke J. H., Carayannopoulos L., Craft C. M., Capra J. D., Tucker P. W. NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss. Mol Cell Biol. 1993 Sep;13(9):5593–5603. doi: 10.1128/mcb.13.9.5593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yang Y. S., Yang M. C., Tucker P. W., Capra J. D. NonO enhances the association of many DNA-binding proteins to their targets. Nucleic Acids Res. 1997 Jun 15;25(12):2284–2292. doi: 10.1093/nar/25.12.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yue Z., Maldonado E., Pillutla R., Cho H., Reinberg D., Shatkin A. J. Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12898–12903. doi: 10.1073/pnas.94.24.12898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yuryev A., Patturajan M., Litingtung Y., Joshi R. V., Gentile C., Gebara M., Corden J. L. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6975–6980. doi: 10.1073/pnas.93.14.6975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zeng C., Berget S. M. Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol Cell Biol. 2000 Nov;20(21):8290–8301. doi: 10.1128/mcb.20.21.8290-8301.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhang W. W., Zhang L. X., Busch R. K., Farrés J., Busch H. Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells. Biochem J. 1993 Feb 15;290(Pt 1):267–272. doi: 10.1042/bj2900267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang Z., Carmichael G. G. The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell. 2001 Aug 24;106(4):465–475. doi: 10.1016/s0092-8674(01)00466-4. [DOI] [PubMed] [Google Scholar]
  65. Zhou Z., Luo M. J., Straesser K., Katahira J., Hurt E., Reed R. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature. 2000 Sep 21;407(6802):401–405. doi: 10.1038/35030160. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES