Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Sep 1;14(17):4336–4349. doi: 10.1002/j.1460-2075.1995.tb00108.x

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

G R Screaton 1, J F Cáceres 1, A Mayeda 1, M V Bell 1, M Plebanski 1, D G Jackson 1, J I Bell 1, A R Krainer 1
PMCID: PMC394518  PMID: 7556075

Abstract

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.

Full text

PDF
4340

Images in this article

4339Image
on p.4339
4341Image
on p.4341
4341Image
on p.4341
4342Image
on p.4342
4343Image
on p.4343
4344Image
on p.4344
4345Image
on p.4345

Selected References

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

  1. Ayane M., Preuss U., Köhler G., Nielsen P. J. A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs. Nucleic Acids Res. 1991 Mar 25;19(6):1273–1278. doi: 10.1093/nar/19.6.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  3. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bossie M. A., DeHoratius C., Barcelo G., Silver P. A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast. Mol Biol Cell. 1992 Aug;3(8):875–893. doi: 10.1091/mbc.3.8.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cardinali B., Cohen P. T., Lamond A. I. Protein phosphatase 1 can modulate alternative 5' splice site selection in a HeLa splicing extract. FEBS Lett. 1994 Oct 3;352(3):276–280. doi: 10.1016/0014-5793(94)00973-2. [DOI] [PubMed] [Google Scholar]
  6. Cavaloc Y., Popielarz M., Fuchs J. P., Gattoni R., Stévenin J. Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family. EMBO J. 1994 Jun 1;13(11):2639–2649. doi: 10.1002/j.1460-2075.1994.tb06554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Champlin D. T., Frasch M., Saumweber H., Lis J. T. Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin. Genes Dev. 1991 Sep;5(9):1611–1621. doi: 10.1101/gad.5.9.1611. [DOI] [PubMed] [Google Scholar]
  8. Cáceres J. F., Krainer A. R. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 1993 Dec;12(12):4715–4726. doi: 10.1002/j.1460-2075.1993.tb06160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  10. Deans J. P., Boyd A. W., Pilarski L. M. Transitions from high to low molecular weight isoforms of CD45 (T200) involve rapid activation of alternate mRNA splicing and slow turnover of surface CD45R. J Immunol. 1989 Aug 15;143(4):1233–1238. [PubMed] [Google Scholar]
  11. Diamond R. H., Du K., Lee V. M., Mohn K. L., Haber B. A., Tewari D. S., Taub R. Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing. J Biol Chem. 1993 Jul 15;268(20):15185–15192. [PubMed] [Google Scholar]
  12. 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]
  13. Fox S. B., Fawcett J., Jackson D. G., Collins I., Gatter K. C., Harris A. L., Gearing A., Simmons D. L. Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res. 1994 Aug 15;54(16):4539–4546. [PubMed] [Google Scholar]
  14. Fu X. D., Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. doi: 10.1126/science.1373910. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Ge H., Zuo P., Manley J. L. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. doi: 10.1016/0092-8674(91)90626-a. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Harper J. E., Manley J. L. Multiple activities of the human splicing factor ASF. Gene Expr. 1992;2(1):19–29. [PMC free article] [PubMed] [Google Scholar]
  21. Jiménez-García L. F., Spector D. L. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell. 1993 Apr 9;73(1):47–59. doi: 10.1016/0092-8674(93)90159-n. [DOI] [PubMed] [Google Scholar]
  22. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  23. Kim Y. J., Zuo P., Manley J. L., Baker B. S. The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2. Genes Dev. 1992 Dec;6(12B):2569–2579. doi: 10.1101/gad.6.12b.2569. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. Lamm G. M., Lamond A. I. Non-snRNP protein splicing factors. Biochim Biophys Acta. 1993 Jun 25;1173(3):247–265. doi: 10.1016/0167-4781(93)90122-t. [DOI] [PubMed] [Google Scholar]
  29. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  30. Mattox W., Ryner L., Baker B. S. Autoregulation and multifunctionality among trans-acting factors that regulate alternative pre-mRNA processing. J Biol Chem. 1992 Sep 25;267(27):19023–19026. [PubMed] [Google Scholar]
  31. Mayeda A., Helfman D. M., Krainer A. R. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. Mol Cell Biol. 1993 May;13(5):2993–3001. doi: 10.1128/mcb.13.5.2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Mayeda A., Munroe S. H., Cáceres J. F., Krainer A. R. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 1994 Nov 15;13(22):5483–5495. doi: 10.1002/j.1460-2075.1994.tb06883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  37. Ring H. Z., Lis J. T. The SR protein B52/SRp55 is essential for Drosophila development. Mol Cell Biol. 1994 Nov;14(11):7499–7506. doi: 10.1128/mcb.14.11.7499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Stephens C., Harlow E. Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins. EMBO J. 1987 Jul;6(7):2027–2035. doi: 10.1002/j.1460-2075.1987.tb02467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  42. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  43. Sureau A., Perbal B. Several mRNAs with variable 3' untranslated regions and different stability encode the human PR264/SC35 splicing factor. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):932–936. doi: 10.1073/pnas.91.3.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tacke R., Boned A., Goridis C. ASF alternative transcripts are highly conserved between mouse and man. Nucleic Acids Res. 1992 Oct 25;20(20):5482–5482. doi: 10.1093/nar/20.20.5482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tanaka M., Herr W. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell. 1990 Feb 9;60(3):375–386. doi: 10.1016/0092-8674(90)90589-7. [DOI] [PubMed] [Google Scholar]
  46. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  47. Treisman R., Orkin S. H., Maniatis T. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes. Nature. 1983 Apr 14;302(5909):591–596. doi: 10.1038/302591a0. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. Zahler A. M., Neugebauer K. M., Stolk J. A., Roth M. B. Human SR proteins and isolation of a cDNA encoding SRp75. Mol Cell Biol. 1993 Jul;13(7):4023–4028. doi: 10.1128/mcb.13.7.4023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zerler B., Moran B., Maruyama K., Moomaw J., Grodzicker T., Ruley H. E. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells. Mol Cell Biol. 1986 Mar;6(3):887–899. doi: 10.1128/mcb.6.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zuo P., Manley J. L. Functional domains of the human splicing factor ASF/SF2. EMBO J. 1993 Dec;12(12):4727–4737. doi: 10.1002/j.1460-2075.1993.tb06161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES