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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 03 May 2017
FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis
Cell Research Open Access 21 November 2014
BMC Systems Biology Open Access 16 July 2012
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Shepard, P.J. & Hertel, K. The SR protein family. Genome Biol. 10, 242 (2009).
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).
Lin, S. & Fu, X.D. SR proteins and related factors in alternative splicing. Adv. Exp. Med. Biol. 623, 107–122 (2007).
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).
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).
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).
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).
Wu, J.Y. & Maniatis, T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75, 1061–1070 (1993).
Blaustein, M. et al. Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT. Nat. Struct. Mol. Biol. 12, 1037–1044 (2005).
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).
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).
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).
Hertel, K.J. Combinatorial control of exon recognition. J. Biol. Chem. 283, 1211–1215 (2008).
Blencowe, B.J. Alternative splicing: new insights from global analyses. Cell 126, 37–47 (2006).
Smith, C.W.J. & Valcarcel, J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000).
Sanford, J.R. et al. Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF. PLoS ONE 3, e3369 (2008).
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).
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).
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).
Ding, J.-H. et al. Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart. EMBO J. 23, 885–896 (2004).
Karni, R. et al. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat. Struct. Mol. Biol. 14, 185–193 (2007).
Ray, D. et al. Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. Nat. Biotechnol. 27, 667–670 (2009).
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).
Sanford, J.R. et al. Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts. Genome Res. 19, 381–394 (2009).
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).
Wei, Y., Harris, T. & Childs, G. Global gene expression patterns during neural differentiation of P19 embryonic carcinoma cells. Differentiation 70, 204–219 (2002).
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).
Liang, H., Tuan, R.S. & Norton, P.A. Overexpression of SR proteins and splice variants modulates chondrogenesis. Exp. Cell Res. 313, 1509–1517 (2007).
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).
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).
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).
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).
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).
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).
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).
Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Penalva, L.O., Tenenbaum, S.A. & Keene, J.D. Gene expression analysis of messenger RNP complexes. Methods Mol. Biol. 257, 125–134 (2004).
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).
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).
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).
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).
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).
Huang, Y. & Steitz, J.A. Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol. Cell 7, 899–905 (2001).
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).
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).
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).
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).
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).
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).
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.).
The authors declare no competing financial interests.
Supplementary Figures 1–10, Supplementary Tables 1, 3 and 4, Supplementary Methods (PDF 718 kb)
RIP hits of SRp20 and SRp75 in undifferentiated P19 cells and cells after 8 days of retinoic acid induction. (PDF 1409 kb)
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)
About this article
Cite this article
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
This article is cited by
Nature Communications (2017)
Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells
FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis
Cell Research (2014)
Nature Reviews Genetics (2013)
Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence