Raw data used in figures.

Calcineurin is a conserved, calcium-regulated phosphatase involved in various functions, including muscle physiology and nervous system activity. The Calcineurin A (CnA) catalytic subunit consists of a phosphatase region and a regulatory domain, which includes a Calcineurin B binding helix, a Calmodulin binding domain, and an auto-inhibitory domain, separated by linker regions (LR1 and LR2). CnA diversity is generated via the existence of paralogous genes and via alternative splicing, producing tissue-specific isoforms, whose functional significance is only partially understood. Our analyses reveal that the LR2 region is an alternative splicing hotspot conserved across paralogous genes and across species, from humans to nematodes. To investigate LR2 variants’ role in vivo, we used the C. elegans model, where a single gene, tax-6, encodes CnA/TAX-6 and produces TAX-6a and TAX-6b/c variants with distinct LR2 regions. TAX-6a is the predominant neuronal isoform, while TAX-6b/c are enriched in muscles. We generated isoform-specific deletions, gain-of-function mutations, and phosphosite mutations using CRISPR/Cas9, and selectively up-regulated Calcineurin signaling in neurons and muscles with transgenes. We quantified behavioral parameters modulated by Calcineurin signaling, such as crawling speed and responses to noxious heat, both as constitutive and experience-dependent traits. Our results show distinct, non-redundant functions for TAX-6a and TAX-6b/c isoforms, synergistic actions across neurons and muscles, and suggest the differential involvement of TAX-6 phosphorylation-based regulation across these tissues in the modulation of phenotypic outcomes. Overall, our study underscores the importance of LR2-affecting splice variants in regulating both constitutive and experience-dependent behaviors and suggests that such tissue-specific regulation might be conserved across species.