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Intron 7 conserved sequence elements regulate the splicing of the SMN genes

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Abstract

Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. In humans there are two nearly identical SMN genes, SMN1 and SMN2. The SMN2 gene generates a truncated protein, due to a C to T nucleotide alteration in exon 7, which leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and the trans-factors that bind them. Here, we identify conserved intronic sequences in the SMN genes. Five regions were examined due to conservation and their proximity to exons 6 through 8. Using mutagenesis two conserved elements located in intron 7 of the SMN genes that affect exon 7 splicing have been identified. Additional analysis of one of these regions showed decreased inclusion of exon 7 in SMN transcripts when deletions or mutations were introduced. Furthermore, multimerization of this conserved region was capable of restoring correct SMN splicing. Together these results describe a novel intronic splicing enhancer sequence located in the final intron of the SMN genes. This discovery provides insight into the splicing of the SMN genes using conserved intonic sequence as a tool to uncover regions of importance in pre-messenger RNA splicing. A better understanding of the way SMN pre-mRNA is spliced can lead to the development of new therapies.

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References

  • Baughan TD, Dickson A et al (2009) Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy. Hum Mol Genet 18(9):1600–1611

    Article  CAS  PubMed  Google Scholar 

  • Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336

    Article  CAS  PubMed  Google Scholar 

  • Cartegni L, Krainer AR (2002) Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30(4):377–384

    Article  CAS  PubMed  Google Scholar 

  • Cartegni L, Wang J et al (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 31(13):3568–3571

    Article  CAS  PubMed  Google Scholar 

  • Chen HH, Chang JG et al (2008) The RNA binding protein hnRNP Q modulates the utilization of exon 7 in the survival motor neuron 2 (SMN2) gene. Mol Cell Biol 28(22):6929–6938

    Article  CAS  PubMed  Google Scholar 

  • Coady TH, Shababi M et al (2007) Restoration of SMN function: delivery of a trans-splicing RNA re-directs SMN2 pre-mRNA splicing. Mol Ther 15(8):1471–1478

    Article  CAS  PubMed  Google Scholar 

  • Coovert DD, Le TT et al (1997) The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet 6(8):1205–1214

    Article  CAS  PubMed  Google Scholar 

  • DiDonato CJ, Chen XN et al (1997) Cloning, characterization, and copy number of the murine survival motor neuron gene: homolog of the spinal muscular atrophy-determining gene. Genome Res 7(4):339–352

    CAS  PubMed  Google Scholar 

  • DiDonato CJ, Lorson CL et al (2001) Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing. Hum Mol Genet 10(23):2727–2736

    Article  CAS  PubMed  Google Scholar 

  • Hofmann Y, Wirth B (2002) hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1. Hum Mol Genet 11(17):2037–2049

    Article  CAS  PubMed  Google Scholar 

  • Hofmann Y, Lorson CL et al (2000) Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc Natl Acad Sci USA 97(17):9618–9623

    Article  CAS  PubMed  Google Scholar 

  • Hua Y, Vickers TA et al (2008) Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am J Hum Genet 82(4):834–848

    Article  CAS  PubMed  Google Scholar 

  • Huang HY, Chien CH et al (2006) RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. Nucleic Acids Res 34(Web Server issue):W429–W434

    Article  CAS  PubMed  Google Scholar 

  • Kashima T, Manley JL (2003) A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet 34(4):460–463

    Article  CAS  PubMed  Google Scholar 

  • Kashima T, Rao N et al (2007) An intronic element contributes to splicing repression in spinal muscular atrophy. Proc Natl Acad Sci USA 104(9):3426–3431

    Article  CAS  PubMed  Google Scholar 

  • Lefebvre S, Burglen L et al (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80(1):155–165

    Article  CAS  PubMed  Google Scholar 

  • Lefebvre S, Burlet P et al (1997) Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 16(3):265–269

    Article  CAS  PubMed  Google Scholar 

  • Lefebvre S, Burglen L et al (1998) The role of the SMN gene in proximal spinal muscular atrophy. Hum Mol Genet 7(10):1531–1536

    Article  CAS  PubMed  Google Scholar 

  • Lorson CL, Hahnen E et al (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA 96(11):6307–6311

    Article  CAS  PubMed  Google Scholar 

  • Miyaso H, Okumura M et al (2003) An intronic splicing enhancer element in survival motor neuron (SMN) pre-mRNA. J Biol Chem 278(18):15825–15831

    Article  CAS  PubMed  Google Scholar 

  • Monani UR, Lorson CL et al (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet 8(7):1177–1183

    Article  CAS  PubMed  Google Scholar 

  • Novelli G, Calza L et al (1997) Expression study of survival motor neuron gene in human fetal tissues. Biochem Mol Med 61(1):102–106

    Article  CAS  PubMed  Google Scholar 

  • Schrank B, Gotz R et al (1997) Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc Natl Acad Sci USA 94(18):9920–9925

    Article  CAS  PubMed  Google Scholar 

  • Singh NK, Singh NN et al (2006) Splicing of a critical exon of human survival motor neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol 26(4):1333–1346

    Article  CAS  PubMed  Google Scholar 

  • Wang W, Furneaux H et al (2000) HuR regulates p21 mRNA stabilization by UV light. Mol Cell Biol 20(3):760–769

    Article  CAS  PubMed  Google Scholar 

  • Young PJ, DiDonato CJ et al (2002) SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2 beta 1. Hum Mol Genet 11(5):577–587

    Article  CAS  PubMed  Google Scholar 

  • Zhang XH, Chasin LA (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18(11):1241–1250

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was generously supported by The Research Institute at Nationwide Children’s Hospital, and the National Institute of Neurological Disorders and Stroke (NINDS) Grant, 1R21NS054690, to DSC, and the National Institute of General Medical Sciences (NIGMS) Grant, 1F31GM080151-01A1, to JTG. We thank Dr. Brian Kaspar for generously provided the NSC-34 cells and Dr. Ravindra Singh for the Casp3Avr minigene.

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Authors and Affiliations

  1. The Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital and the Department of Pediatrics and Integrated Biomedical Science Graduate Program (IBGP), The Ohio State University, 700 Children’s Drive, Columbus, OH, 43205, USA

    Jordan T. Gladman & Dawn S. Chandler

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  1. Jordan T. Gladman
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  2. Dawn S. Chandler
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Correspondence to Dawn S. Chandler.

Electronic supplementary material

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439_2009_733_MOESM1_ESM.eps

Supplemental Fig. 1 Conservation alignment of the SMN genes. The SMN genes of 17 different vertebrate and invertebrate animals were compared using the 2004 UCSC genome browser. Areas of high conservation are represented by the height of the bar graph at the top of the diagram. The red boxes highlight the areas of conservation that were examined in closer detail. (EPS 916 kb)

439_2009_733_MOESM2_ESM.doc

Supplemental Table 1 Mutagenesis primers used to create the SMN mutations outlined in this paper. Only the sense strand for each primer pair is shown. (DOC 33 kb)

439_2009_733_MOESM3_ESM.doc

Supplemental Table 2 Multispecies alignment of conserved sequence I6-1. The sequences are shown in the reverse complement orientation as depicted using UCSG Genome Browser March 2006 genomic assembly (DOC 29 kb)

439_2009_733_MOESM4_ESM.doc

Supplemental Table 3 Multispecies alignment of conserved sequence I6-2. The sequences are shown in the reverse complement orientation as depicted using UCSG Genome Browser March 2006 genomic assembly. (DOC 40 kb)

439_2009_733_MOESM5_ESM.doc

Supplemental Table 4 Multispecies alignment of conserved sequence I6-3. The sequences are shown in the reverse complement orientation as depicted using UCSG Genome Browser March 2006 genomic assembly. (DOC 29 kb)

439_2009_733_MOESM7_ESM.doc

Supplemental Table 5 Multispecies alignment of conserved sequence I7-1. The sequences are shown in the reverse complement orientation as depicted using UCSG Genome Browser March 2006 genomic assembly. (DOC 29 kb)

439_2009_733_MOESM8_ESM.doc

Supplemental Table 6 Multispecies alignment of conserved sequence I7-2. The sequences are shown in the reverse complement orientation as depicted using UCSG Genome Browser March 2006 genomic assembly. (DOC 31 kb)

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Gladman, J.T., Chandler, D.S. Intron 7 conserved sequence elements regulate the splicing of the SMN genes. Hum Genet 126, 833–841 (2009). https://doi.org/10.1007/s00439-009-0733-7

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  • DOI: https://doi.org/10.1007/s00439-009-0733-7

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