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Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells

Nature Structural & Molecular Biology volume 17, pages 962–970 (2010)Cite this article

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Abstract

Members of the SR protein family of RNA-binding proteins have numerous roles in mRNA metabolism, from transcription to translation. To understand how SR proteins coordinate gene regulation, comprehensive knowledge of endogenous mRNA targets is needed. Here we establish physiological expression of GFP-tagged SR proteins from stable transgenes. Using the GFP tag for immunopurification of mRNPs, mRNA targets of SRp20 and SRp75 were identified in cycling and neurally induced P19 cells. Genome-wide analysis showed that SRp20 and SRp75 associate with hundreds of distinct, functionally related groups of transcripts that change in response to neural differentiation. Knockdown of either SRp20 or SRp75 led to up- or downregulation of specific transcripts, including identified targets, and rescue by the GFP-tagged SR proteins proved their functionality. Thus, SR proteins contribute to the execution of gene-expression programs through their association with distinct endogenous mRNAs.

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Figure 1: Expression of endogenous and GFP-tagged SRp20 and SRp75 in P19 cells during neural differentiation.
Figure 2: Total SR protein expression is not increased in BAC transgenic cell lines.
Figure 3: Changes in gene expression upon differentiation are linked to SR protein function.
Figure 4: RIP-chip analysis of SRp20- and SRp75-associated mRNAs in undifferentiated and neural P19 cells.
Figure 5: SRp20 and SRp75 associate with distinct sets of mRNPs that depend on the cell type.
Figure 6: Expression of SRp20 and SRp75 RIP hits is compromised by SR protein depletion.
Figure 7: Misexpression of RIP hits upon depletion of SR proteins and rescue by GFP-tagged SR proteins expressed from BACs.
Figure 8: SR protein depletion leads to misregulation of neural gene expression.

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References

  1. Shepard, P.J. & Hertel, K. The SR protein family. Genome Biol. 10, 242 (2009).

    Article  Google Scholar 

  2. Zhong, X.-Y., Wang, P., Han, J., Rosenfeld, M.G. & Fu, X.-D. SR proteins in vertical integration of gene expression from transcription to RNA processing to translation. Mol. Cell 35, 1–10 (2009).

    Article  CAS  Google Scholar 

  3. Lin, S. & Fu, X.D. SR proteins and related factors in alternative splicing. Adv. Exp. Med. Biol. 623, 107–122 (2007).

    Article  Google Scholar 

  4. Shen, H., Kan, J.L.C. & Green, M.R. Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly. Mol. Cell 13, 367–376 (2004).

    Article  CAS  Google Scholar 

  5. Sapra, A.K. et al. SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol. Cell 34, 179–190 (2009).

    Article  CAS  Google Scholar 

  6. Mayeda, A., Screaton, G.R., Chandler, S.D., Fu, X.-D. & Krainer, A.R. Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements. Mol. Cell. Biol. 19, 1853–1863 (1999).

    Article  CAS  Google Scholar 

  7. Caceres, J.F., Misteli, T., Screaton, G.R., Spector, D.L. & Krainer, A.R. Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J. Cell Biol. 138, 225–238 (1997).

    Article  CAS  Google Scholar 

  8. Wu, J.Y. & Maniatis, T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75, 1061–1070 (1993).

    Article  CAS  Google Scholar 

  9. Blaustein, M. et al. Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT. Nat. Struct. Mol. Biol. 12, 1037–1044 (2005).

    Article  CAS  Google Scholar 

  10. Sanford, J.R., Ellis, J.D., Cazalla, D. & Caceres, J.F. Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor. Proc. Natl. Acad. Sci. USA 102, 15042–15047 (2005).

    Article  CAS  Google Scholar 

  11. Hanamura, A., Caceres, J.F., Mayeda, A., Franza, B.R. Jr. & Krainer, A.R. Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. RNA 4, 430–444 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Zahler, A.M., Neugebauer, K.M., Lane, W.S. & Roth, M.B. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science 260, 219–222 (1993).

    Article  CAS  Google Scholar 

  13. Hertel, K.J. Combinatorial control of exon recognition. J. Biol. Chem. 283, 1211–1215 (2008).

    Article  CAS  Google Scholar 

  14. Blencowe, B.J. Alternative splicing: new insights from global analyses. Cell 126, 37–47 (2006).

    Article  CAS  Google Scholar 

  15. Smith, C.W.J. & Valcarcel, J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000).

    Article  CAS  Google Scholar 

  16. Sanford, J.R. et al. Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF. PLoS ONE 3, e3369 (2008).

    Article  Google Scholar 

  17. Merz, C., Urlaub, H., Will, C.L. & Luhrmann, R. Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment. RNA 13, 116–128 (2007).

    Article  CAS  Google Scholar 

  18. Caceres, J.F., Screaton, G.R. & Krainer, A.R. A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes Dev. 12, 55–66 (1998).

    Article  CAS  Google Scholar 

  19. Gabut, M., Dejardin, J., Tazi, J. & Soret, J. The SR family proteins B52 and dASF/SF2 modulate development of the Drosophila visual system by regulating specific RNA targets. Mol. Cell. Biol. 27, 3087–3097 (2007).

    Article  CAS  Google Scholar 

  20. Ding, J.-H. et al. Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart. EMBO J. 23, 885–896 (2004).

    Article  CAS  Google Scholar 

  21. Karni, R. et al. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat. Struct. Mol. Biol. 14, 185–193 (2007).

    Article  CAS  Google Scholar 

  22. Ray, D. et al. Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. Nat. Biotechnol. 27, 667–670 (2009).

    Article  CAS  Google Scholar 

  23. Wang, J., Smith, P.J., Krainer, A.R. & Zhang, M.Q. Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes. Nucleic Acids Res. 33, 5053–5062 (2005).

    Article  CAS  Google Scholar 

  24. Sanford, J.R. et al. Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts. Genome Res. 19, 381–394 (2009).

    Article  CAS  Google Scholar 

  25. McBurney, M.W., Jones-Villeneuve, E.M., Edwards, M.K. & Anderson, P.J. Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature 299, 165–167 (1982).

    Article  CAS  Google Scholar 

  26. Wei, Y., Harris, T. & Childs, G. Global gene expression patterns during neural differentiation of P19 embryonic carcinoma cells. Differentiation 70, 204–219 (2002).

    Article  CAS  Google Scholar 

  27. 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. 13, 4023–4028 (1993).

    Article  CAS  Google Scholar 

  28. Liang, H., Tuan, R.S. & Norton, P.A. Overexpression of SR proteins and splice variants modulates chondrogenesis. Exp. Cell Res. 313, 1509–1517 (2007).

    Article  CAS  Google Scholar 

  29. Faa, V. et al. Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. J. Biol. Chem. 284, 30024–30031 (2009).

    Article  CAS  Google Scholar 

  30. Ramchatesingh, J., Zahler, A.M., Neugebauer, K.M., Roth, M.B. & Cooper, T.A. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. Mol. Cell. Biol. 15, 4898–4907 (1995).

    Article  CAS  Google Scholar 

  31. Exline, C.M., Feng, Z. & Stoltzfus, C.M. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 Vif gene expression. J. Virol. 82, 3921–3931 (2008).

    Article  CAS  Google Scholar 

  32. Galiana-Arnoux, D. et al. The CD44 Alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20. J. Biol. Chem. 278, 32943–32953 (2003).

    Article  CAS  Google Scholar 

  33. Lim, L.P. & Sharp, P.A. Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats. Mol. Cell. Biol. 18, 3900–3906 (1998).

    Article  CAS  Google Scholar 

  34. Jumaa, H. & Nielsen, P. The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation. EMBO J. 16, 5077–5085 (1997).

    Article  CAS  Google Scholar 

  35. Lou, H., Neugebauer, K.M., Gagel, R.F. & Berget, S.M. Regulation of alternative polyadenylation by U1 snRNPs and SRp20. Mol. Cell. Biol. 18, 4977–4985 (1998).

    Article  CAS  Google Scholar 

  36. Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

    Article  CAS  Google Scholar 

  37. 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. 115, 587–596 (1991).

    Article  CAS  Google Scholar 

  38. Neugebauer, K.M. & Roth, M.B. Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription. Genes Dev. 11, 1148–1159 (1997).

    Article  CAS  Google Scholar 

  39. Sun, S., Zhang, Z., Sinha, R., Karni, R. & Krainer, A.R. SF2/ASF autoregulation involves multiple layers of post-transcriptional and translational control. Nat. Struct. Mol. Biol. 17, 306–312 (2010).

    Article  CAS  Google Scholar 

  40. Ni, J.Z. et al. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev. 21, 708–718 (2007).

    Article  CAS  Google Scholar 

  41. Lareau, L.F., Inada, M., Green, R.E., Wengrod, J.C. & Brenner, S.E. Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature 446, 926–929 (2007).

    Article  CAS  Google Scholar 

  42. Thomas, P.D. et al. Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools. Nucleic Acids Res. 34, W645–650 (2006).

    Article  Google Scholar 

  43. Schaal, T.D. & Maniatis, T. Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences. Mol. Cell. Biol. 19, 1705–1719 (1999).

    Article  CAS  Google Scholar 

  44. Cavaloc, Y., Bourgeois, C.F., Kister, L. & Stevenin, J. The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. RNA 5, 468–483 (1999).

    Article  CAS  Google Scholar 

  45. Zheng, Z.M., He, P.J. & Baker, C.C. Structural, functional, and protein binding analyses of bovine papillomavirus type 1 exonic splicing enhancers. J. Virol. 71, 9096–9107 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Kittler, R. et al. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat. Methods 4, 337–344 (2007).

    Article  CAS  Google Scholar 

  47. Penalva, L.O., Tenenbaum, S.A. & Keene, J.D. Gene expression analysis of messenger RNP complexes. Methods Mol. Biol. 257, 125–134 (2004).

    CAS  PubMed  Google Scholar 

  48. Hogan, D.J., Riordan, D.P., Gerber, A.P., Herschlag, D. & Brown, P.O. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 6, e255 (2008).

    Article  Google Scholar 

  49. Gama-Carvalho, M., Barbosa-Morais, N., Brodsky, A., Silver, P. & Carmo-Fonseca, M. Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors. Genome Biol. 7, R113 (2006).

    Article  Google Scholar 

  50. Hieronymus, H. & Silver, P.A. Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery. Nat. Genet. 33, 155–161 (2003).

    Article  CAS  Google Scholar 

  51. Bjork, P. et al. Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions. J. Cell Biol. 184, 555–568 (2009).

    Article  Google Scholar 

  52. Talavera, D., Orozco, M. & de la Cruz, X. Alternative splicing of transcription factors' genes: beyond the increase of proteome diversity. Comp. Funct. Genomics doi:10.1155/2009/905894 (published online 12 July 2009).

  53. Huang, Y. & Steitz, J.A. Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol. Cell 7, 899–905 (2001).

    Article  CAS  Google Scholar 

  54. Huang, Y., Gattoni, R., Stévenin, J. & Steitz, J.A. SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol. Cell 11, 837–843 (2003).

    Article  CAS  Google Scholar 

  55. Blanchette, M., Labourier, E., Green, R.E., Brenner, S.E. & Rio, D.C. Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs. Mol. Cell 14, 775–786 (2004).

    Article  CAS  Google Scholar 

  56. Boutz, P.L. et al. A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons. Genes Dev. 21, 1636–1652 (2007).

    Article  CAS  Google Scholar 

  57. Sanford, J.R., Gray, N.K., Beckmann, K. & Caceres, J.F. A novel role for shuttling SR proteins in mRNA translation. Genes Dev. 18, 755–768 (2004).

    Article  CAS  Google Scholar 

  58. Henschel, A., Buchholz, F. & Habermann, B. DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113–W120 (2004).

    Article  CAS  Google Scholar 

  59. Kittler, R., Heninger, A.-K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat. Methods 2, 779–784 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Ule and C. Smith for helpful comments on the manuscript, J. Jarrells and B. Jedamzik for performing the microarray experiments and B. Habermann and J. Howard for valuable advice on the analysis of the RIP-chip data. The financial support was from the Sigrid Juselius foundation (to M.-L.Ä.), the Helsingin Sanomain Foundation (to M.-L.Ä.), the Max Planck Society (to K.M.N.) and the European Commission (EURASNET-518238 to K.M.N.).

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Authors and Affiliations

  1. Max Planck Institute of Cell Biology and Genetics, Dresden, Germany

    Minna-Liisa Änkö, Lucia Morales, Ian Henry & Karla M Neugebauer

  2. Biotechnology Center, Technische Universität Dresden, Dresden, Germany

    Andreas Beyer

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  1. Minna-Liisa Änkö
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  2. Lucia Morales
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  3. Ian Henry
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Contributions

M.-L.Ä. and K.M.N. designed the experiments; M.-L.Ä. conducted the experiments; A.B. and L.M. designed the RIP-chip data analysis; I.H., L.M. and M.-L.Ä. performed the data analysis; M.-L.Ä. and K.M.N. wrote the manuscript.

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Correspondence to Karla M Neugebauer.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1, 3 and 4, Supplementary Methods (PDF 718 kb)

Supplementary Table 2

RIP hits of SRp20 and SRp75 in undifferentiated P19 cells and cells after 8 days of retinoic acid induction. (PDF 1409 kb)

Supplementary Table 5

List of genes that changed more than 1.5-fold in expression upon SRp75 or SRp20 knockdown compared to the control (p-value<0.05, one-way ANOVA). (PDF 131 kb)

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Änkö, ML., Morales, L., Henry, I. et al. Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells. Nat Struct Mol Biol 17, 962–970 (2010). https://doi.org/10.1038/nsmb.1862

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