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. 1992 Oct 25;20(20):5263–5269. doi: 10.1093/nar/20.20.5263

Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing.

J E Mermoud 1, P Cohen 1, A I Lamond 1
PMCID: PMC334330  PMID: 1331983

Abstract

We have used a combination of highly specific protein phosphatase inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spliceosome assembly. Okadaic acid, tautomycin, and microcystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an important role in regulating the pre-mRNA splicing mechanism.

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Selected References

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

  1. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  2. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brautigan D. L., Shriner C. L. Methods to distinguish various types of protein phosphatase activity. Methods Enzymol. 1988;159:339–346. doi: 10.1016/0076-6879(88)59034-1. [DOI] [PubMed] [Google Scholar]
  4. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  5. Cohen P. Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts. Methods Enzymol. 1991;201:389–398. doi: 10.1016/0076-6879(91)01035-z. [DOI] [PubMed] [Google Scholar]
  6. Cohen P., Foulkes J. G., Holmes C. F., Nimmo G. A., Tonks N. K. Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 1988;159:427–437. doi: 10.1016/0076-6879(88)59042-0. [DOI] [PubMed] [Google Scholar]
  7. Cohen P., Holmes C. F., Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci. 1990 Mar;15(3):98–102. doi: 10.1016/0968-0004(90)90192-e. [DOI] [PubMed] [Google Scholar]
  8. Cohen P., Klumpp S., Schelling D. L. An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 1989 Jul 3;250(2):596–600. doi: 10.1016/0014-5793(89)80803-8. [DOI] [PubMed] [Google Scholar]
  9. Cohen P. Protein phosphorylation and hormone action. Proc R Soc Lond B Biol Sci. 1988 Jul 22;234(1275):115–144. doi: 10.1098/rspb.1988.0040. [DOI] [PubMed] [Google Scholar]
  10. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [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. 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]
  13. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  14. Hernandez N., Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. doi: 10.1016/0092-8674(83)90211-8. [DOI] [PubMed] [Google Scholar]
  15. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  16. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  17. Kuret J., Bell H., Cohen P. Identification of high levels of protein phosphatase-1 in rat liver nuclei. FEBS Lett. 1986 Jul 28;203(2):197–202. doi: 10.1016/0014-5793(86)80741-4. [DOI] [PubMed] [Google Scholar]
  18. Lamond A. I., Konarska M. M., Sharp P. A. A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev. 1987 Aug;1(6):532–543. doi: 10.1101/gad.1.6.532. [DOI] [PubMed] [Google Scholar]
  19. Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
  20. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  21. MacKintosh C., Beattie K. A., Klumpp S., Cohen P., Codd G. A. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 1990 May 21;264(2):187–192. doi: 10.1016/0014-5793(90)80245-e. [DOI] [PubMed] [Google Scholar]
  22. MacKintosh C., Klumpp S. Tautomycin from the bacterium Streptomyces verticillatus. Another potent and specific inhibitor of protein phosphatases 1 and 2A. FEBS Lett. 1990 Dec 17;277(1-2):137–140. doi: 10.1016/0014-5793(90)80828-7. [DOI] [PubMed] [Google Scholar]
  23. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  24. Padgett R. A., Hardy S. F., Sharp P. A. Splicing of adenovirus RNA in a cell-free transcription system. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5230–5234. doi: 10.1073/pnas.80.17.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  26. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  27. Ryder U., Sproat B. S., Lamond A. I. Sequence-specific affinity selection of mammalian splicing complexes. Nucleic Acids Res. 1990 Dec 25;18(24):7373–7379. doi: 10.1093/nar/18.24.7373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sola M. M., Langan T., Cohen P. p34cdc2 phosphorylation sites in histone H1 are dephosphorylated by protein phosphatase 2A1. Biochim Biophys Acta. 1991 Sep 3;1094(2):211–216. doi: 10.1016/0167-4889(91)90011-l. [DOI] [PubMed] [Google Scholar]
  29. Tazi J., Daugeron M. C., Cathala G., Brunel C., Jeanteur P. Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing. J Biol Chem. 1992 Mar 5;267(7):4322–4326. [PubMed] [Google Scholar]
  30. Turcq B., Dobinson K. F., Serizawa N., Lambowitz A. M. A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1676–1680. doi: 10.1073/pnas.89.5.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wooley J. C., Zukerberg L. R., Chung S. Y. Polypeptide components of human small nuclear ribonucleoproteins. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5208–5212. doi: 10.1073/pnas.80.17.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woppmann A., Patschinsky T., Bringmann P., Godt F., Lührmann R. Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants. Nucleic Acids Res. 1990 Aug 11;18(15):4427–4438. doi: 10.1093/nar/18.15.4427. [DOI] [PMC free article] [PubMed] [Google Scholar]

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