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Piccolo paralogs and orthologs display conserved patterns of alternative splicing within the C2A and C2B domains

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Abstract

Piccolo is an organizational component of the presynaptic active zone, a specialized region of nerve terminals where synaptic vesicles fuse and release their neurotransmitter contents. Alternative splicing (AS) of the mouse Piccolo gene (PCLO) produces two primary splice isoforms: isoform-1 that includes two C2 domains (C2A and C2B) and isoform-2 with only C2A. Genome-wide association studies have identified variations located in or near the C2A domain of human Piccolo that predispose individuals to affective disorders and in rare cases leads to altered brain development. In zebrafish a genome duplication event led to the generation of PCLO-a and PCLO-b: gene paralogs that display strikingly similar genomic organization with other PCLO orthologs. Given this conservation in genomic structure, it is likely that AS patterns of zebrafish PCLO paralogs are similar to mammalian PCLO. We used a RT-PCR strategy to identify four zebrafish isoforms generated from zebrafish PCLO-a and PCLO-b that are equivalent to mouse Piccolo isoform-1 and isoform-2. Additionally, we identified an exon skipping event that leads to exclusion of a 27 nucleotide exon in both zebrafish Piccolo-a and Piccolo-b. Elimination of this exon in mammalian Piccolo alters the calcium binding property of the C2A domain. We also measured transcriptional levels of mouse and zebrafish Piccolo splice variants and demonstrate that despite similarities in AS, there are quantitative differences in gene expression. Our results indicate that AS of Piccolo is similar across diverse taxa and further support the use of zebrafish to study the role of Piccolo in neurodevelopment and synaptic signaling.

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Acknowledgments

This study was supported by start-up funds to S. Fenster from Fort Lewis College, an undergraduate research grant from Fort Lewis College to D. Fountain and an undergraduate research grant from Ashland University to L. Knapp. The authors thank Catherine P. Fenster for helpful comments and suggestions on the manuscript.

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    1. Department of Biology, Fort Lewis College, 1000 Rim Drive, Durango, CO, 81301, USA

      David I. Fountain & Steven D. Fenster

    2. Ashland University, Ashland, OH, USA

      Lindsey Knapp & Mason Posner

    3. San Juan College, Farmington, NM, USA

      Keith Baugh

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    1. David I. Fountain
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    2. Lindsey Knapp
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    Correspondence to Steven D. Fenster.

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    The respective Animal Use Care Committees at Ashland University and Fort Lewis College approved all animal procedures.

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    David I. Fountain and Lindsey Knapp have contributed equally to this work.

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    Fountain, D.I., Knapp, L., Baugh, K. et al. Piccolo paralogs and orthologs display conserved patterns of alternative splicing within the C2A and C2B domains. Genes Genom 38, 407–419 (2016). https://doi.org/10.1007/s13258-015-0383-1

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