Poster abstracts

Poster number 34 submitted by Fran Jodelka

Identification of novel regulators of SMN2 exon 7 splicing in spinal muscular atrophy

Francine M. Jodelka (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.), Claribel D Wee (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.), Michelle L Hastings (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.)

Abstract:
       Promoting SMN2 exon 7 inclusion is a promising therapeutic strategy for SMA. Some members of the SR protein family of splicing factors have been found to control alternative splicing of exon 7. However, a comprehensive survey of all SR proteins in the context of exon 7 splicing has not been performed.

       We knocked down each member of the SR protein family (SRSF 1-12) independently in cells through transfection of siRNAs. In a reciprocal experiment, individual SR proteins were overexpressed in cells by transfection of cloned, human cDNAs into HeLa and HEK293-T cells. Changes in exon 7 inclusion in SMN2-derived transcripts were quantitated by RT-PCR and PAGE. SR proteins that were found to inhibit exon 7 inclusion were knocked down in human SMA patient cell lines and the effect on SMN protein levels was assessed by western blot.

       Knock down of individual SR proteins revealed novel regulators of SMN2 alternative splicing. Lowered expression of some proteins correlates with an increase in exon 7 inclusion while lowered expression of other members of the SR protein family correlates with a reduction in inclusion. Overexpression of these proteins confirmed that modulation of exon 7 inclusion was due to specific SR protein abundance. Knock-down of inhibitors of exon 7 inclusion resulted in an increase in SMN protein in SMA patient fibroblasts.

       SR proteins are known to influence alternative splicing, and in this family, we have identified a number of novel regulators of SMN exon 7 inclusion. SR proteins may be promising candidates for targeted inhibition; there is a high level of redundancy amongst spliceosome components; they have sequence-specific binding activities, and therefore, SR protein bioactivity can be potentially modified with high degrees of specificity and sensitivity. Consequently, manipulating SR protein activity is a viable therapeutic approach to SMA that could pose a reduced risk of treatment toxicity.

Keywords: SMN2, SMA, splicing