Cell-Specific RNA Binding Protein Rbfox2 Regulates Ca_V2.2 mRNA Exon Composition and Ca_V2.2 Current Size

The majority of multiexon mammalian genes contain alternatively spliced exons that have unique expression patterns in different cell populations and that have important cell functions. The expression profiles of alternative exons are controlled by cell-specific splicing factors that can promote exon inclusion or exon skipping but with few exceptions we do not know which specific splicing factors control the expression of alternatively spliced exons of known biological function. Many ion channel genes undergo extensive alternative splicing including Cacna1b that encodes the voltage-gated Ca_V2.2 α1 subunit. Alternatively spliced exon 18a in Cacna1b RNA encodes 21 amino acids in the II-III loop of Ca_V2.2, and its expression differs across the nervous system and over development. Genome-wide, protein-RNA binding analyses coupled to high-throughput RNA sequencing show that RNA binding Fox (Rbfox) proteins associate with Ca_V2.2 (Cacna1b) pre-mRNAs. Here, we link Rbfox2 to suppression of e18a. We show increased e18a inclusion in Ca_V2.2 mRNAs: (1) after siRNA knockdown of Rbfox2 in a neuronal cell line and (2) in RNA from sympathetic neurons of adult compared to early postnatal mice. By immunoprecipitation of Rbfox2-RNA complexes followed by qPCR, we demonstrate reduced Rbfox2 binding upstream of e18a in RNA from sympathetic neurons of adult compared to early postnatal mice. Ca_V2.2 currents in cell lines and in sympathetic neurons expressing only e18a-Ca_V2.2 are larger compared to currents from those expressing only Δ18a-Ca_V2.2. We conclude that Rbfox2 represses e18a inclusion during pre-mRNA splicing of Ca_V2.2, limiting the size of Ca_V2.2 currents early in development in certain neuronal populations.