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. 1996 Aug 20;93(17):8830–8835. doi: 10.1073/pnas.93.17.8830

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

S Yitzhaki 1, E Miriami 1, R Sperling 1, J Sperling 1
PMCID: PMC38553  PMID: 8799112

Abstract

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

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Selected References

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

  1. Ast G., Goldblatt D., Offen D., Sperling J., Sperling R. A novel splicing factor is an integral component of 200S large nuclear ribonucleoprotein (InRNP) particles. EMBO J. 1991 Feb;10(2):425–432. doi: 10.1002/j.1460-2075.1991.tb07964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Crispino J. D., Blencowe B. J., Sharp P. A. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994 Sep 23;265(5180):1866–1869. doi: 10.1126/science.8091213. [DOI] [PubMed] [Google Scholar]
  3. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fu X. D., Mayeda A., Maniatis T., Krainer A. R. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11224–11228. doi: 10.1073/pnas.89.23.11224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  7. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  8. García-Blanco M. A., Jamison S. F., Sharp P. A. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. doi: 10.1101/gad.3.12a.1874. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Ghetti A., Piñol-Roma S., Michael W. M., Morandi C., Dreyfuss G. hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 1992 Jul 25;20(14):3671–3678. doi: 10.1093/nar/20.14.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gui J. F., Lane W. S., Fu X. D. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 1994 Jun 23;369(6482):678–682. doi: 10.1038/369678a0. [DOI] [PubMed] [Google Scholar]
  12. Jamison S. F., Pasman Z., Wang J., Will C., Lührmann R., Manley J. L., Garcia-Blanco M. A. U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements. Nucleic Acids Res. 1995 Aug 25;23(16):3260–3267. doi: 10.1093/nar/23.16.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  14. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  15. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  16. Lin C. H., Patton J. G. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995 May;1(3):234–245. [PMC free article] [PubMed] [Google Scholar]
  17. Madhani H. D., Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. doi: 10.1146/annurev.ge.28.120194.000245. [DOI] [PubMed] [Google Scholar]
  18. Mayeda A., Zahler A. M., Krainer A. R., Roth M. B. Two members of a conserved family of nuclear phosphoproteins are involved in pre-mRNA splicing. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1301–1304. doi: 10.1073/pnas.89.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mermoud J. E., Cohen P. T., Lamond A. I. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J. 1994 Dec 1;13(23):5679–5688. doi: 10.1002/j.1460-2075.1994.tb06906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miriami E., Angenitzki M., Sperling R., Sperling J. Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles. J Mol Biol. 1995 Feb 17;246(2):254–263. doi: 10.1006/jmbi.1994.0081. [DOI] [PubMed] [Google Scholar]
  21. Miriami E., Sperling J., Sperling R. Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles. Nucleic Acids Res. 1994 Aug 11;22(15):3084–3091. doi: 10.1093/nar/22.15.3084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roscigno R. F., Garcia-Blanco M. A. SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA. 1995 Sep;1(7):692–706. [PMC free article] [PubMed] [Google Scholar]
  23. Roth M. B., Murphy C., Gall J. G. A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle. J Cell Biol. 1990 Dec;111(6 Pt 1):2217–2223. doi: 10.1083/jcb.111.6.2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  26. Spann P., Feinerman M., Sperling J., Sperling R. Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs. Proc Natl Acad Sci U S A. 1989 Jan;86(2):466–470. doi: 10.1073/pnas.86.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sperling R., Spann P., Offen D., Sperling J. U1, U2, and U6 small nuclear ribonucleoproteins (snRNPs) are associated with large nuclear RNP particles containing transcripts of an amplified gene in vivo. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6721–6725. doi: 10.1073/pnas.83.18.6721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sperling R., Sperling J., Levine A. D., Spann P., Stark G. R., Kornberg R. D. Abundant nuclear ribonucleoprotein form of CAD RNA. Mol Cell Biol. 1985 Mar;5(3):569–575. doi: 10.1128/mcb.5.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tarn W. Y., Steitz J. A. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994 Nov 15;8(22):2704–2717. doi: 10.1101/gad.8.22.2704. [DOI] [PubMed] [Google Scholar]
  31. Tazi J., Kornstädt U., Rossi F., Jeanteur P., Cathala G., Brunel C., Lührmann R. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 1993 May 20;363(6426):283–286. doi: 10.1038/363283a0. [DOI] [PubMed] [Google Scholar]
  32. Vellard M., Sureau A., Soret J., Martinerie C., Perbal B. A potential splicing factor is encoded by the opposite strand of the trans-spliced c-myb exon. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2511–2515. doi: 10.1073/pnas.89.7.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  34. Wu Z. A., Murphy C., Callan H. G., Gall J. G. Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes. J Cell Biol. 1991 May;113(3):465–483. doi: 10.1083/jcb.113.3.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zachar Z., Kramer J., Mims I. P., Bingham P. M. Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans. J Cell Biol. 1993 May;121(4):729–742. doi: 10.1083/jcb.121.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  37. Zahler A. M., Neugebauer K. M., Lane W. S., Roth M. B. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science. 1993 Apr 9;260(5105):219–222. doi: 10.1126/science.8385799. [DOI] [PubMed] [Google Scholar]
  38. Zahler A. M., Roth M. B. Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2642–2646. doi: 10.1073/pnas.92.7.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zamore P. D., Green M. R. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991 Jan;10(1):207–214. doi: 10.1002/j.1460-2075.1991.tb07937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  41. Zhang G., Taneja K. L., Singer R. H., Green M. R. Localization of pre-mRNA splicing in mammalian nuclei. Nature. 1994 Dec 22;372(6508):809–812. doi: 10.1038/372809a0. [DOI] [PubMed] [Google Scholar]
  42. Zhang M., Zamore P. D., Carmo-Fonseca M., Lamond A. I., Green M. R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769–8773. doi: 10.1073/pnas.89.18.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]

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