SR proteins regulate V6 exon splicing of CD44 pre-mRNA

  • Loh, Tiing Jen (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Moon, Heegyum (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Jang, Ha Na (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Liu, Yongchao (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Choi, Namjeong (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Shen, Shengfu (Willston Northampton School) ;
  • Williams, Darren Reece (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Jung, Da-Woon (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Zheng, Xuexiu (School of life Sciences, Gwangju Institute of Science and Technology) ;
  • Shen, Haihong (School of life Sciences, Gwangju Institute of Science and Technology)
  • Received : 2016.07.21
  • Accepted : 2016.08.16
  • Published : 2016.11.30


CD44 pre-mRNA includes 20 exons, of which exons 1-5 ($C_1-C_5$) and exons 16-20 ($C_6-C_{10}$) are constant exons, whereas exons 6-15 ($V_1-V_{10}$) are variant exons. $V_6$-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 $V_6$ splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 $V_6$ minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect $V_6$ splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit $V_6$ splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a $V_6$ specific primer, we identified that reduced SRSF2 expression significantly reduced the $V_6$ isoform, but increased $V_{6-10}$ and $V_{6,8-10}$ isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 $V_6$ splicing.


CD44;Pre-mRNA splicing;SR protein;$V_6$ exon


Supported by : National Research Foundation (NRF) of Korea, Gwangju Institute of Science and Technology (GIST)


  1. Green MR (1986) Pre-mrna splicing. Annu Rev Genet 20, 671-708
  2. David CJ and Manley JL (2010) Alternative pre-mRNA splicing regulation in cancer: Pathways and programs unhinged. Genes Dev 24, 2343-2364
  3. Cooper TA, Wan L and Dreyfuss G (2009) Rna and disease. Cell 136, 777-793
  4. Kim E, Goren A and Ast G (2008) Insights into the connection between cancer and alternative splicing. Trends Genet 24, 7-10
  5. Busch A and Hertel KJ (2012) Evolution of sr protein and hnrnp splicing regulatory factors. Wiley Interdiscip Rev RNA 3, 1-12
  6. Han SP, Tang YH and Smith R (2010) Functional diversity of the hnrnps: Past, present and perspectives. Biochem J 430, 379-392
  7. Herrlich P, Morrison H, Sleeman J et al (2000) Cd44 acts both as a growth-and invasiveness-promoting molecule and as a tumor-suppressing cofactor. Ann N Y Acad Sci 910, 106-118; discussion 118-120
  8. Screaton GR, Bell MV, Bell JI and Jackson DG (1993) The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse pgp-1 (cd44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat. J Biol Chem 268, 12235-12238
  9. Bennett KL, Jackson DG, Simon JC et al (1995) Cd44 isoforms containing exon v3 are responsible for the presentation of heparin-binding growth factor. J Cell Biol 128, 687-698
  10. Orian-Rousseau V and Ponta H (2008) Adhesion proteins meet receptors: A common theme? Adv Cancer Res 101, 63-92
  11. Tremmel M, Matzke A, Albrecht I et al (2009) A cd44v6 peptide reveals a role of cd44 in vegfr-2 signaling and angiogenesis. Blood 114, 5236-5244
  12. Orian-Rousseau V, Chen L, Sleeman JP, Herrlich P and Ponta H (2002) Cd44 is required for two consecutive steps in hgf/c-met signaling. Genes Dev 16, 3074-3086
  13. Legg JW and Isacke CM (1998) Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, cd44. Curr Biol 8, 705-708
  14. Boukis LA, Liu N, Furuyama S and Bruzik JP (2004) Ser/arg-rich protein-mediated communication between u1 and u2 small nuclear ribonucleoprotein particles. J Biol Chem 279, 29647-29653
  15. Shao W, Kim HS, Cao Y, Xu YZ and Query CC (2012) A u1-u2 snrnp interaction network during intron definition. Mol Cell Biol 32, 470-478
  16. Bai Y, Lee D, Yu T and Chasin LA (1999) Control of 3' splice site choice in vivo by asf/sf2 and hnrnp a1. Nucleic Acids Res 27, 1126-1134
  17. Zhong XY, Wang P, Han J, Rosenfeld MG and Fu XD (2009) Sr proteins in vertical integration of gene expression from transcription to RNA processing to translation. Mol Cell 35, 1-10
  18. Loh TJ, Moon H, Cho S et al (2014) Sc35 promotes splicing of the c5-v6-c6 isoform of cd44 pre-mRNA. Oncol Rep 31, 273-279
  19. Mi Y, Zhang C, Bu Y et al (2015) DEPDC1 is a novel cell cycle related gene that regulates mitotic progression. BMB Rep 48, 413-418
  20. Han A, Chae YC, Park JW, Kim KB, Kim JY, Seo SB (2015) Transcriptional repression of ANGPT1 by histone H3K9 demethylase KDM3B. BMB Rep 48, 401-406
  21. Sun Q, Mayeda A, Hampson RK, Krainer AR and Rottman FM (1993) General splicing factor sf2/asf promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev 7, 2598-2608
  22. Chandler SD, Mayeda A, Yeakley JM, Krainer AR and Fu XD (1997) Rna splicing specificity determined by the coordinated action of RNA recognition motifs in sr proteins. Proc Natl Acad Sci U S A 94, 3596-3601
  23. Anko ML, Morales L, Henry I, Beyer A and Neugebauer KM (2010) Global analysis reveals srp20-and srp75-specific mrnps in cycling and neural cells. Nat Struct Mol Biol 17, 962-970
  24. Pandit S, Zhou Y, Shiue L et al (2013) Genome-wide analysis reveals sr protein cooperation and competition in regulated splicing. Mol Cell 50, 223-235
  25. Simard MJ and Chabot B (2002) Srp30c is a repressor of 3' splice site utilization. Mol Cell Biol 22, 4001-4010
  26. Han J, Ding JH, Byeon CW et al (2011) Sr proteins induce alternative exon skipping through their activities on the flanking constitutive exons. Mol Cell Biol 31, 793-802
  27. Erkelenz S, Mueller WF, Evans MS et al (2013) Position-dependent splicing activation and repression by sr and hnrnp proteins rely on common mechanisms. RNA 19, 96-102
  28. Bielli P, Bordi M, Di Biasio V and Sette C (2014) Regulation of bcl-x splicing reveals a role for the polypyrimidine tract binding protein (ptbp1/hnrnp i) in alternative 5' splice site selection. Nucleic Acids Res 42, 12070-12081
  29. van Weering DH, Baas PD and Bos JL (1993) A PCR-based method for the analysis of human cd44 splice products. PCR Methods Appl 3, 100-106

Cited by

  1. Influence of transcriptional variants on metastasis pp.1555-8584, 2018,