Characterising the transcriptional profile of CACNA1C expression in rodents

Introduction: The CACNA1C gene, which codes for the ɑ1-subunit of the voltage-gated calcium channel Cav1.2, has been repeatedly associated with psychiatric disease in genome-wide studies. Cavv1.2 can be expressed in a number of isoforms produced through alternative splicing of pre-mRNA. Splice variants in CACNA1C have been shown to be spatially and temporally regulated, many variants being tissue specific or changing expression during development. Moreover, CACNA1C splice variants are known to have differing electrophysiological properties as well as differences in expression. With the advent of long-read sequencing technologies, it has become possible to reliably identify and characterise mRNA splice variants, and recent evidence from human post-mortem brains suggests that the transcriptional profile of CACNA1C may be under-characterised in rodents. In this project, we have set out to fully characterise the full range of mRNA splice variants in mice and rats across neural and cardiovascular tissues. Methods: We extracted whole-tissue RNA from aorta, heart, cerebellum, and frontal cortex of 8 mice and 8 rats. Full-length CACNA1C cDNA was isolated from each tissue using RT and PCR amplification of transcripts starting with exon1a, exon1b, or exon1c. Sequencing libraries were prepared as per the Nanopore 1D Ligation Sequencing protocol, and sequenced on the Nanopore MinION. Transcripts were then translated using the online ExPaSy tool and aligned on UniProt to annotate and predict functional significance of individual splice variants. Results: Principal component analysis (PCA) of sequencing reads reveals a clear segregation of transcript isoforms enriched in the brain compared to isoforms enriched in the heart and isoforms enriched in smooth muscle tissue. Across all samples, reads showed 53 different transcript variants, out of which 45 novel splice variants, among which 37 are predicted to be coding, and 3 which contain novel previously uncharacterised exons. Conclusion: The transcriptional profile of CACNA1C in rodents has been under-characterised. Although our results need to be validated at the proteomic level, we reveal 45 novel mRNA splice variants. Further study of these may reveal insights to the pathophysiology of psychiatric disorders associated with CACNA1C, and potentially lead to novel therapeutic targets.