Abstract
Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.
Highlights
Calcium is a prevalent second messenger controlling many cellular functions and playing a critical role in health and disease [1,2,3]
By manipulating the endogenous alternative promoters and exons of unc-68 with CRISPR/Cas9-mediated editing, we demonstrate their utility in modulating many biological functions such as pharyngeal pumping, swimming and crawling behaviors
Two alternative transcriptional start sites leading to the expression of two mutually exclusive first exons have been defined for unc-68, but nothing is known about isoform expression patterns
Summary
Calcium is a prevalent second messenger controlling many cellular functions and playing a critical role in health and disease [1,2,3]. Intracellular calcium signals are used to couple stimuli with cell activity. The Ryanodine Receptors (RyRs) are calcium-activated calcium channels expressed at the membrane of the endoplasmic/sarcoplasmic reticulum (ER/ SR)[4]. One of their functions is to amplify intracellular calcium signals in the cytosol by mobilizing intracellular stores from the ER/SR, in a process called Calcium-Induced Calcium Release (CICR)[5]. The three RyR proteins are close homologs, with about 65% sequence identity [8] Their sequences differ the most in three divergent regions named DR1, DR2 and DR3 [7, 9]
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