REGULATION OF SMN2 ALTERNATIVE SPLICING BY THE RNA-BINDING PROTEIN SAM68 AND ITS IMPLICATION IN THE RECOVERY OF SMN PROTEIN IN SMA CELLS

Most human genes contain introns that need to be excised from the mRNA before its translation through a process named splicing. The regulation of splicing is orchestrated by numerous proteins, RNAs and sequence elements located in the introns and exons of the gene that compose the so called "splicing code". Up to 50-60% of disease-causing genetic mutations are thought to affect the splicing code without altering the open reading frames of the proteins encoded by the gene. One of the best characterized genetic diseases caused by such mutations is Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder representing the primary genetic cause of infant mortality. SMA is caused by inactivating mutations in SMN1, which encodes the Survival of Motor Neuron protein (SMN). Although SMA patients retain the almost identical SMN2 gene, a single nucleotide change in SMN2 causes the exclusion of exon 7 from the mature mRNA and the production of an unstable protein. Hence, regulation of exon 7 splicing in the SMN2 mRNA represents a valuable therapeutic approach for the correction of this disease-causing genetic defect in SMA patients. We have identified Sam68 as a splicing factor that causes exon 7 skipping in SMN2 and we have shown that it cooperates with hnRNP A1 in the regulation of this event. Inhibition of Sam68 function in SMA fibroblasts restores exon 7 inclusion and SMN protein expression. The proposed project aims at defining the mechanisms by which Sam68 regulates the alternative splicing of SMN2 and at developing reagents and tools that interfere with Sam68 function in vivo and can rescue SMN function in SMA cells. The results obtainable with this project are relevant for the comprehension of the molecular mechanisms involved in the defect causing SMA, a well described example of inheritable disease of known genetic cause, and might hold promise for future applications as therapeutic approaches to SMA. Furthermore, since defects in splicing are likely to affect other neurodegenerative diseases whose cause remains unknown, our studies might provide a useful model also for the studies and the understanding of such diseases.