Many biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 SRSF6-target genes in skin and show that this SR-rich protein binds to alternative exons in the pre-mRNA of the extracellular-matrix protein tenascin C, thus promoting the expression of isoforms characteristic of invasive and metastatic cancer independently of cell type. SRSF6 overexpression additionally results in depletion of LGR6+ stem cells and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the tenascin-C isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis.
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Krainer, A.R., Conway, G.C. & Kozak, D. The essential pre-mRNA splicing factor SF2 influences 5′ splice site selection by activating proximal sites. Cell 62, 35–42 (1990).
Ge, H. & Manley, J.L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell 62, 25–34 (1990).
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. 21, 5803–5816 (1993).
Srebrow, A. & Kornblihtt, A.R. The connection between splicing and cancer. J. Cell Sci. 119, 2635–2641 (2006).
Ritchie, W., Granjeaud, S., Puthier, D. & Gautheret, D. Entropy measures quantify global splicing disorders in cancer. PLoS Comput. Biol. 4, e1000011 (2008).
Karni, R. et al. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat. Struct. Mol. Biol. 14, 185–193 (2007).
Anczuków, O. et al. The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation. Nat. Struct. Mol. Biol. 19, 220–228 (2012).
Jia, R., Li, C., McCoy, J.P., Deng, C.X. & Zheng, Z.M. SRp20 is a proto-oncogene critical for cell proliferation and tumor induction and maintenance. Int. J. Biol. Sci. 6, 806–826 (2010).
Ghigna, C. et al. Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol. Cell 20, 881–890 (2005).
Dvorak, H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986).
Dunham, L.J. Cancer in man at site of prior benign lesion of skin or mucous membrane: a review. Cancer Res. 32, 1359–1374 (1972).
Schäfer, M. & Werner, S. Cancer as an overhealing wound: an old hypothesis revisited. Nat. Rev. Mol. Cell Biol. 9, 628–638 (2008).
Singer, A.J. & Clark, R.A. Cutaneous wound healing. N. Engl. J. Med. 341, 738–746 (1999).
Ffrench-Constant, C., Van de Water, L., Dvorak, H.F. & Hynes, R.O. Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J. Cell Biol. 109, 903–914 (1989).
McJunkin, K. et al. Reversible suppression of an essential gene in adult mice using transgenic RNA interference. Proc. Natl. Acad. Sci. USA 108, 7113–7118 (2011).
Paladini, R.D., Takahashi, K., Bravo, N.S. & Coulombe, P.A. Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16. J. Cell Biol. 132, 381–397 (1996).
Chiquet-Ehrismann, R., Mackie, E.J., Pearson, C.A. & Sakakura, T. Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 47, 131–139 (1986).
Midwood, K.S. & Orend, G. The role of tenascin-C in tissue injury and tumorigenesis. J. Cell Commun. Signal. 3, 287–310 (2009).
Fuchs, E. The tortoise and the hair: slow-cycling cells in the stem cell race. Cell 137, 811–819 (2009).
Stenn, K.S. & Paus, R. Controls of hair follicle cycling. Physiol. Rev. 81, 449–494 (2001).
Snippert, H.J. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010).
Waikel, R.L., Kawachi, Y., Waikel, P.A., Wang, X.J. & Roop, D.R. Deregulated expression of c-Myc depletes epidermal stem cells. Nat. Genet. 28, 165–168 (2001).
Kaur, P., Li, A., Redvers, R. & Bertoncello, I. Keratinocyte stem cell assays: an evolving science. J. Investig. Dermatol. Symp. Proc. 9, 238–247 (2004).
Salomonis, N. et al. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors. PLoS Comput. Biol. 5, e1000553 (2009).
Cartegni, L., Chew, S.L. & Krainer, A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).
Bailey, T.L. & Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36 (1994).
Liu, H.X., Zhang, M. & Krainer, A.R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12, 1998–2012 (1998).
Clower, C.V. et al. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc. Natl. Acad. Sci. USA 107, 1894–1899 (2010).
Wang, Z. et al. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons. J. Mol. Cell Biol. 4, 79–87 (2012).
David, C.J., Chen, M., Assanah, M., Canoll, P. & Manley, J.L. HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature 463, 364–368 (2010).
Orend, G. & Chiquet-Ehrismann, R. Tenascin-C induced signaling in cancer. Cancer Lett. 244, 143–163 (2006).
Akerman, M., David-Eden, H., Pinter, R.Y. & Mandel-Gutfreund, Y. A computational approach for genome-wide mapping of splicing factor binding sites. Genome Biol. 10, R30 (2009).
Fukunaga-Kalabis, M. et al. Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population. Oncogene 29, 6115–6124 (2010).
Tsunoda, T. et al. Involvement of large tenascin-C splice variants in breast cancer progression. Am. J. Pathol. 162, 1857–1867 (2003).
Morasso, M.I. & Tomic-Canic, M. Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing. Biol. Cell 97, 173–183 (2005).
Kasper, M. et al. Wounding enhances epidermal tumorigenesis by recruiting hair follicle keratinocytes. Proc. Natl. Acad. Sci. USA 108, 4099–4104 (2011).
Christofk, H.R. et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230–233 (2008).
Yagi, H. et al. HNK-1 epitope-carrying tenascin-C spliced variant regulates the proliferation of mouse embryonic neural stem cells. J. Biol. Chem. 285, 37293–37301 (2010).
Dang, C. et al. Tenascin-C patterns and splice variants in actinic keratosis and cutaneous squamous cell carcinoma. Br. J. Dermatol. 155, 763–770 (2006).
Guttery, D.S. et al. Association of invasion-promoting tenascin-C additional domains with breast cancers in young women. Breast Cancer Res. 12, R57 (2010).
Esposito, I. et al. Tenascin C and annexin II expression in the process of pancreatic carcinogenesis. J. Pathol. 208, 673–685 (2006).
Schliemann, C. et al. Three clinical-stage tumor targeting antibodies reveal differential expression of oncofetal fibronectin and tenascin-C isoforms in human lymphoma. Leuk. Res. 33, 1718–1722 (2009).
Frey, K. et al. Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions. Exp. Dermatol. 20, 685–688 (2011).
Takeda, A. et al. Plasma large Tenascin-C spliced variant as a possible biomarker for the prediction of hepatic recurrence in colorectal cancer. Surgery 141, 124–125 (2007).
Takeda, A. et al. Clinical significance of large tenascin-C spliced variant as a potential biomarker for colorectal cancer. World J. Surg. 31, 388–394 (2007).
Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005).
Premsrirut, P.K. et al. A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell 145, 145–158 (2011).
Aaboe, M. et al. SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization. Cancer Res. 66, 3434–3442 (2006).
Saldanha, A.J. Java Treeview: extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
Huang, D.W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
Lampugnani, M.G. Cell migration into a wounded area in vitro. Methods Mol. Biol. 96, 177–182 (1999).
McFarland, H.I. & Rosenberg, A.S. Skin allograft rejection. Curr. Protoc. Immunol. 84, 4.4 (2009).
Crooks, G.E., Hon, G., Chandonia, J.M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Shapiro, M.B. & Senapathy, P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15, 7155–7174 (1987).
Carmel, I., Tal, S., Vig, I. & Ast, G. Comparative analysis detects dependencies among the 5′ splice-site positions. RNA 10, 828–840 (2004).
Sanford, J.R. et al. Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF. PLoS ONE 3, e3369 (2008).
Fregoso, O.I., Das, S., Akerman, M. & Krainer, A.R. Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces cellular senescence. Mol. Cell 50, 56–66 (2013).
Hanamura, A., Cáceres, 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).
We thank R. Jaenisch (Whitehead Institute) for KH2 ES cells; C. Miething and S.W. Lowe (Memorial Sloan-Kettering Cancer Center) for the targeting construct; P. Premsrirut for useful discussions and help with production of stable embryonic stem-cell lines; S.Y. Kim from the Gene Targeting Shared Resource (Cold Spring Harbor Laboratory) for help with tetraploid complementation procedures; C. Johns for support with microarray analysis; M. Motley for assistance with RT-PCR analysis; and L. Chartarifsky for tissue-microarray staining. We thank L. Bianco for help with animal procedures. We are grateful to B. Boettner, M. Egeblad and A. Mills for critical comments on the manuscript. This study was supported by grant CA13107 from the US National Cancer Institute and was performed with assistance from Cold Spring Harbor Laboratory Shared Resources funded in part by Cancer Center Support grant 5P30CA045508. M.A.J. was supported by a Danish Cancer Society postdoctoral fellowship.
The authors declare no competing financial interests.
Integrated supplementary information
(a) A human SRSF6 cDNA (with an N-terminal T7-tag) was inserted into pBS31'-RBGpA-TREtight-ColA1 flp-in. Upon induction, the cDNA is expressed as a bicistronic transcript, including an IRES element followed by EGFP cDNA. The reverse tetracycline-controlled transactivator (M2-rtTA) is expressed from the Rosa26 locus in KH2 ES cells. (b) Expression of ectopic SRSF6 (T7) and GFP protein was measured by immunoblotting using antibodies against the T7-tag or GFP upon DOX treatment of R26-rtTA/ColA1-SRSF6-transgenic mice. Protein was extracted from thymus (th), liver (li), skin (sk), small intestine (in), brain (br), heart (he), kidney (ki), and spleen (sp). Loading: 0.1% Ponceau S staining. (c) Sporadic expression of ectopic SRSF6 in bone marrow. Control (left panel), +DOX (right panel). T7-tag antibody detects ectopically expressed SRSF6. Bar = 100 μm (upper panel), 20 μm (lower panel).
(a) Epidermis from transgenic mouse not exposed to doxycycline. Note continuous basal layer, 1-2 cell thick spinous layer, and thin stratum corneum (bar = 50 μm). (b) Normal epidermis 200 μm from area of hyperplasia (bar = 50 μm). (c) Overexpression of SRSF6 for 7 days leads to mild epidermal hyperplasia. Note the orthokeratotic hyperkeratotic (arrow) stratum corneum. The granular layer is also more prominent (short arrow) (bar = 50 μm). (d) Prominent thickened stratum spinosum (arrow) and minimal dysplasia in mild early lesion (bar = 50 μm). (e) Overexpression of SRSF6 for 14 days results in severe hyperplasia. Note the severe hyperplasia in the epidermis and hair follicles and thick parakeratotic hyperkeratotic crust (arrow) (bar = 100 μm). (f) Higher magnification of e. There is severe hyperplasia of the epidermis (arrow) and hair follicles (short arrow) (bar = 100 μm).
(a) Intra-epidermal microabscess (short arrow) and mild inflammation (arrow) at the disturbed dermal-epidermal interface. (bar = 100 μm). (b) Multiple apoptotic bodies (arrows) and dyskeratosis (faulty development of the epidermis with abnormal keratinization) (short arrow) (bar = 100 μm). (c) Spongiosis (loss of adhesion and intercellular edema) in the epidermis (bar = 100 μm). (d) Basal cell apoptosis (Civatte body) (arrow), spongiosis, and dyskeratosis (bar = 100 μm).
(a) Severe epidermal hyperplasia (short arrow) and exorbitant parakeratotic hyperkeratosis (arrow) (bar = 100 μm). (b) Dyskeratosis with severe parakeratosis (arrows). Hair follicles (short arrows) are trapped in the rapidly proliferating hyperplastic epidermis (bar = 50 μm). (c) Thickened epidermis (dotted line) with extensive hyperkeratosis and keratin pearls trapped in the hyperplastic stratum corneum (arrow) (bar = 50 μm). (d) Severe dysplasia with severe dyskeratosis, excessive apoptotic bodies (arrows), and dysplastic keratinocytes (short arrows) (bar = 25 μm).
Supplementary Figure 5 RT-PCR validation of differentially expressed genes upon SRSF6 overexpression in skin.
14 out of 14 genes tested were successfully validated by RT-PCR. Confirmed upregulated genes include: genes induced upon tissue injury, such as Keratin 6 (Krt6) (microarray: +154-fold), Keratin 16 (Krt16) (microarray: +13-fold) and tenascin C (Tnc) (microarray: +6-fold); and pro-inflammatory cytokines important for wound healing, such as tumor necrosis factor (Tnf) (microarray: +6-fold), Interleukin-1 b (Il1b) (microarray: +24-fold), Chemokine (C-X-C motif) ligand 2 (Cxcl2) (microarray: +97-fold), Chemokine (C-C motif) ligand 3 (Ccl3) (microarray: +43-fold). Confirmed downregulated genes included: genes related to skin stem cells, such as Keratin 15 (Krt15) (microarray: -5-fold), Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) (microarray: -5-fold); and genes related to differentiation, such as Filaggrin-2 (Flg2) (microarray: -4-fold) and Stearoyl-coenzyme A desaturase 3 (Scd3) (microarray: -18-fold).
Primary keratinocytes from control (R26-rtTA) or double-transgenic (R26-rtTA/ColA1-SRSF6) mice were allowed to differentiate in the presence of 1.2 mM Ca2+ for 5 d. (a) Induction of SRSF6 by DOX-treatment (+) strongly promoted differentiation of primary compared to control keratinocytes, as seen by the strong induction of genes associated with keratinocyte differentiation, measured by RT-qPCR: loricrin (Lor: +4-fold), involucrin (Inv: +7-fold), keratin 1 (Krt1: +743-fold), filaggrin (Flg: +32-fold). In contrast, genes associated with undifferentiated keratinocytes, such as keratin 5 (Krt5: +3-fold) and keratin 14 (Krt14: +2-fold) were relatively unchanged upon SRSF6 induction. (b) The corresponding increase in loricrin protein levels upon SRSF6 induction was validated by immunoblotting; no change was observed in DOX-treated control keratinocytes (note: loading was different than for double-transgenic keratinocytes). Keratin 5 protein levels were unaffected by DOX treatment in both control and double-transgenic keratinocytes.
Validation of SRSF6-responsive ASEs using 32P-radioactive RT-PCR. (a) Strong ASEs with significant change; *p<0.05: Phosphatidylinositol transfer protein, cytoplasmic 1 (Pitpnc1), kinesin family member 20B (Kif20b), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (Magi1), synaptotagmin VIII (Syt8), hexokinase 3 (Hk3), SH2-domain-containing 5 (Sh2d5), lines homolog (Drosophila)(Lins2), ankyrin 1, erythrocytic (Ank1), ETS-domain protein (SRF accessory protein 1) (Elk4), Pyruvate Kinase 2 (Pkm2). (b) Weak ASEs that were not significant: tryptase alpha/beta (Tpsab1), growth factor receptor-bound protein 7 (Grb7). (c) ASEs that did not show anticipated splicing changes by RT-PCR and therefore could not be validated: brain-specific angiogenesis inhibitor 2 (Bai2), valosin containing protein (Vcp), family with sequence similarity 160, member B2 (Fam160b2), armadillo-repeat gene deleted in velocardiofacial syndrome (Arvcf). Statistical analysis employed a Mann-Whitney test (n=10). Data are represented as mean +/- s.e.m. (d) Tnc splicing changes upon Srsf6 knockdown in NIH-3T3 cells. 32P-radioactive RT-PCR validation shows reciprocal splicing changes in Tnc upon Srsf6 knockdown, using primers specific for Tnc exons 9 and 16. Tnc +E10-15 and ΔE14 isoforms decreased upon Srsf6 depletion, whereas Tnc-S (ΔE10-15) (constitutive isoform) was unaffected. Lanes shown correspond to two biological replicates. (e) Pictogram of the predicted SRSF6 in vivo binding motif (9-mer; CCWKSWGSM, Top) which shows similarity to the reported SRSF6 functional SELEX binding motif (6-mer; YRCRKM, Bottom). The central 5-mer of the predicted SRSF6 in vivo binding motif is denoted ‘5-mer core motif’.
(a) Human A2058 melanoma cells transduced with inducible short-hairpins against either TNC or SRSF6 were used for wound-healing assays. Cells were treated with DOX (10 μg/ml) for 4 d in total. Cell migration was measured over 18 h. Knockdown of either SRSF6 or TNC impairs wound healing (2 shRNAs per gene). (b) Immunoblotting confirms efficient SRSF6 knockdown. NSC=no-silencing control.
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Jensen, M., Wilkinson, J. & Krainer, A. Splicing factor SRSF6 promotes hyperplasia of sensitized skin. Nat Struct Mol Biol 21, 189–197 (2014). https://doi.org/10.1038/nsmb.2756
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