Alternative splicing regulatory mechanisms of hnRNP C in Spinal Muscular Atrophy

Abstract

Alternative splicing (AS) generates more than one mRNA isoforms from a single gene. Spinal muscular atrophy (SMA) is an autosomal recessive disorder with progressive muscle weakness and atrophy. SMA occurs by bi-allelic and recessive deletions or mutations in the survival motor neuron 1 (SMN1) gene that causes reduced expression of essential survival motor neuron (SMN) protein. Humans have a paralogous gene of SMN1, called SMN2, which have mutations to cause skipping of exon 7 and further produce ~90% of non-functional SMN protein. Through shRNA-mediated knockdown (KD) and overexpression of hnRNP C, I found here that hnRNP C inhibits SMN2 cassette exon 7 splicing. I further identified novel AS targets of hnRNP C using RNA-mediated oligonucleotide Annealing, Selection, and Ligation with next-generation sequencing (RASL-seq). Gene Ontology (GO) analysis demonstrate that the genes with altered AS by hnRNP C include functions in various neurodegenerative diseases. I finally identified hnRNP C functional target sequence that is located at 24 nucleotides (nt) upstream from the 3’ splice-site of cassette exon in SMN2 pre-mRNA by using mutagenesis assay of SMN2 minigene and RNA-pulldown immunoblotting assay of synthetic biotin-labeled RNAs. In conclusion, hnRNP C regulates SMN2 cassette exon splicing through interaction with its upstream intron.