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

Abstract

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 CaV2.2 α1 subunit. Alternatively spliced exon 18a in Cacna1b RNA encodes 21 amino acids in the II-III loop of CaV2.2, 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 CaV2.2 (Cacna1b) pre-mRNAs. Here, we link Rbfox2 to suppression of e18a. We show increased e18a inclusion in CaV2.2 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. CaV2.2 currents in cell lines and in sympathetic neurons expressing only e18a-CaV2.2 are larger compared to currents from those expressing only Δ18a-CaV2.2. We conclude that Rbfox2 represses e18a inclusion during pre-mRNA splicing of CaV2.2, limiting the size of CaV2.2 currents early in development in certain neuronal populations.

Tissue‐ and cell‐specific expression of a splice variant in the II‐III cytoplasmic loop of Cacna1b

“…In this mouse model, DNA sequences between e18 and e19 (e18‐e18a intron, e18a, and e18a‐e19 intron) were removed using homologous recombination (Fig. A, ). To confirm deletion of e18a sequence, we performed RT‐PCR in whole‐brain samples from WT and ∆e18a‐only mice.…”

“…In the adult nervous system, the abundance of +e18a‐ Cacna1b mRNA differs among tissues. Higher levels of +e18a‐ Cacna1b mRNA are observed in dorsal root ganglia, superior cervical ganglia, and spinal cord relative to whole brain . Interestingly, the relative abundance between +e18a‐ Cacna1b and ∆e18a‐ Cacna1b mRNAs also differs among brain subregions.…”

“…Functionally, +e18a‐Ca V 2.2 channels are more resistant to cumulative inactivation induced by repetitive stimulation and closed‐state inactivation than ∆e18a‐Ca V 2.2 channels . Furthermore, +e18a‐Ca V 2.2 channels exhibit larger calcium currents relative to ∆e18a‐Ca V 2.2 channels in both mammalian expression systems and neurons . Despite all of this information, very little is known about the functional role of +18a‐ Cacna1b and ∆e18a‐ Cacna1b splice variants in the nervous system.…”

Tissue and cell-type specific expression of a splice variant in the II-III cytoplasmic loop ofCacna1b

“…In the adult nervous system, the abundance of +e18a-Cacna1b mRNA differs among tissues. Higher levels of +e18a-Cacna1b mRNA are observed in dorsal root ganglia, superior cervical ganglia, and spinal cord relative to whole brain [6,16]. Interestingly, the relative abundance between +e18a-Cacna1b and De18a-Cacna1b mRNAs also differs among brain subregions.…”

“…Functionally, +e18a-CaV2.2 channels are more resistant to cumulative inactivation induced by repetitive stimulation and closedstate inactivation than De18a-CaV2.2 channels [9]. Furthermore, +e18a-CaV2.2 channels exhibit larger calcium currents relative to De18a-CaV2.2 channels in both mammalian expression systems and neurons [16]. Despite all of this information, very little is known about the functional role of +18a-Cacna1b and De18a-Cacna1b splice variants in the nervous system.…”