Spinal muscular atrophy (SMA) results from the loss of both telomeric 5q13 copies of the SMN1 gene. The centromeric 5q13 SMN2 gene encodes an identical protein. While the loss of SMN2 does not lead to the development of SMA, the presence of SMN2 acts as a disease modifier in a dose-dependent manner in the absence of SMN1. The SMN1 gene produces a full-length transcript, while the primary product of SMN2 is an exon-skipped mRNA lacking exon 7. We reported that a single non-polymorphic nucleotide difference between SMN1 and SMN2 is the basis for this alternative splicing. By comparing hybrid SMN genes derived from SMA patients with unaffected relatives, we demonstrated that the origin of this nucleotide (SMN1 or SMN2) dictated clinical outcome. Therefore, the clinical evolution of SMA correlates with the splicing of SMN genes. SMN protein self-associates, and the protein translated from the alternative spliced SMN2 RNA, has a reduced ability to form oligomers and is less-stable compared to the full-length protein. Furthermore, SMN point mutants from SMA patients show defective oligomerization proportional to their disease severity. These studies have defined the genetic and biochemical basis for development of SMA. The primary defect of the centromeric SMN2 copy gene is the production of the exon 7 skipped SMN protein. Since the """"""""inclusion"""""""" of this exon has been shown to be perhaps the most critical molecular determinant in SMA pathogenesis, the focus of this proposal will be on the determinants that govern exon 7 splicing. The goals of Aims 1 and 2 are to identify the cis-elements that regulate SMN exon 7 RNA-processing, including exonic splice enhancers, silencers, flanking intronic elements, and the critical C/T transition within exon 7 (SMN1=C; SMN2=T). This will be accomplished by extensive genetic analysis and complemented by in vitro analysis of splicing. Extending the genetic work developed in the first two aims, Aim 3 will identify and characterize the trans factors that mediate proper processing of SMN exon 7 pre-mRNA. It will be determined whether factor binding to exon 7 correlates with the ability to stimulate exon 7 inclusion. These studies may be useful for the development of therapeutics that promote the inclusion of exon 7 in SMN2-derived mRNAs.
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