Intron 7 conserved sequence elements regulate the splicing of the SMN genes
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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.
KeywordsSpinal Muscular Atrophy Survival Motor Neuron SMN2 Gene Survival Motor Neuron Gene Splice Element
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.