Regulation of SMN2 Splicing by Inhibition of Protein Phospatase 1 Activity

Specific Aims One way to stop the loss of motorneurons in spinal muscular atrophy would be to influence the splicing patterns of SMN2 exon 7 towards exon inclusion. Inclusion of exon 7 is promoted by the splicing factor tra2-betal, a member of the SR protein family. We showed that protein phosphatase 1 (PP!) binds to the RNA recognition motif of tra2­ betal, which in turn inhibits RNA binding and dephosphorylates tra2-betal. The nuclear inhibitor of PPl, NIPPl, is a protein that binds to PPl and inactivates it. NIPPl and chemical substances that inhibit PPl promote exon 7 inclusion in vivo. Valproic acid is an histone deacetylase inhibitor that promotes exon 7 inclusion by an as yet unknown mechanism. Valproic acid causes the degradation of HDAC2. HDAC2 binds to NIPPl and HDAC2 proteolysis caused by valproic acid increased the amount of NIPPl bound to PPl. This suggests that valproic acid acts though NIPPl to inhibit PPl, which promotes exon inclusion. Neither NIPPl, nor chemical inhibitors of PPl show selectivity towards exon 7 inclusion. In a col1aboration between and organic chemist (David S. Watt) and a molecular biologist (Stefan Stamm) we generated chimeric compounds composed of an oligonucleotide targeting exon 7 and a PPl inhibiting cantharidin moiety. The compounds enter the cel1, strongly promote exon 7 inclusion and can be easily chemical1y modified into tools to analyze exon 7 enhancer complexes. The overall goal of the application is to use the principle of PPl inhibition to selectively increase exon 7 inclusion. This goal will be achieved by pursuing two specific aims: 1) Test the hypothesis that valproic acid promotes exon 7 inclusion by degrading HDAC2, which releases NIPP1 that sequesters PP1 and activates the binding oftra2-beta1 to SMN exon 7 RNA; 2) Optimize chimeric compounds consisting of PP1 inhibiting cantharidin derivatives and oligonucleotides (SMODT compounds) for highest exon 7 inclusion and selectivity;