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Human Genetics

, 126:833 | Cite as

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

  • Jordan T. Gladman
  • Dawn S. ChandlerEmail author
Original Investigation

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.

Keywords

Spinal Muscular Atrophy Survival Motor Neuron SMN2 Gene Survival Motor Neuron Gene Splice Element 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

Supplementary material

439_2009_733_MOESM1_ESM.eps (916 kb)
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 (34 kb)
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 (29 kb)
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 (40 kb)
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 (29 kb)
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 (29 kb)
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 (31 kb)
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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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 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 UniversityColumbusUSA

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