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

Area of Science:

• Molecular Biology
• Neuroscience
• Genetics

Background:

• Alternative splicing of multiexon mammalian genes generates diverse protein isoforms with cell-specific functions.
• Splicing factors controlling alternative exon expression remain largely unknown, hindering understanding of gene regulation.
• The Cacna1b gene, encoding the Ca_V2.2 channel α1 subunit, undergoes extensive alternative splicing, including exon 18a, with differential expression across the nervous system and development.

Purpose of the Study:

• To identify the splicing factors regulating alternative exon 18a inclusion in Cacna1b pre-mRNAs.
• To elucidate the functional consequences of alternative splicing of exon 18a on Ca_V2.2 channel activity.

Main Methods:

• Genome-wide RNA-binding protein analyses and high-throughput RNA sequencing to identify RNA binding Fox (Rbfox) proteins interacting with Cacna1b pre-mRNAs.
• siRNA knockdown of Rbfox2 in neuronal cell lines to assess its effect on e18a inclusion.
• Analysis of e18a inclusion and Rbfox2 binding in sympathetic neurons from mice at different developmental stages.
• Quantitative PCR (qPCR) to measure Rbfox2 binding to Cacna1b pre-mRNA.
• Electrophysiological recordings to compare Ca_V2.2 channel currents with and without e18a inclusion.

Main Results:

• Rbfox proteins, specifically Rbfox2, were found to associate with Cacna1b pre-mRNAs.
• Knockdown of Rbfox2 increased e18a inclusion in Cacna1b mRNAs in neuronal cell lines.
• Sympathetic neurons from adult mice showed lower e18a inclusion and reduced Rbfox2 binding upstream of e18a compared to early postnatal mice.
• Ca_V2.2 channels incorporating e18a exhibited larger currents than those lacking it.

Conclusions:

• Rbfox2 acts as a repressor of e18a inclusion during Cacna1b pre-mRNA splicing.
• Rbfox2-mediated repression of e18a limits the size of Ca_V2.2 currents in specific neuronal populations during early development.