Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo

Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0021, USA.
International Journal of Developmental Neuroscience (Impact Factor: 2.58). 06/2002; 20(3-5):161-71. DOI: 10.1016/S0736-5748(02)00046-1
Source: PubMed


Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation.

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Available from: Steven D Fenster
    • "Generation of I-1 results from an alternative splice donor (ASD) site in exon 20 that leads to removal by splicing of an in-frame stop codon found in the shorter I-2 (Fig. 1a). Deduced amino acid sequence analysis indicated that the 5 additional exons included in I-1 (exons 21-25) are translated into a second predicted C2 domain (C2B) in addition to the single C2A domain found in I-2 (Wang et al. 1999; Fenster and Garner 2002). Although both I-1 and I-2 have been detected in adult rat and mouse brain, comparison and quantification of gene expression levels between these major splice variants has not been examined. "
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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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    • "For Piccolino down regulation in rod photoreceptors, we used an shRNA, which was previously shown to successfully down regulate all Piccolo isoforms in cultured hippocampal neurons when introduced via lentiviral transduction (Pclo28; Leal-Ortiz et al., 2008; Waites et al., 2011). The target sequence for Pclo28 is located in Exon 1 of Piccolo, which encodes segments of the full-length Piccolo as well as the C-terminally truncated, ribbon synapse specific Piccolino (Figure 1A; Fenster and Garner, 2002; Regus-Leidig et al., 2013). To interfere with Piccolo/Piccolino "
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    ABSTRACT: Piccolo is the largest known cytomatrix protein at active zones of chemical synapses. A growing number of studies on conventional chemical synapses assign Piccolo a role in the recruitment and integration of molecules relevant for both endo- and exocytosis of synaptic vesicles, the dynamic assembly of presynaptic F-actin, as well as the proteostasis of presynaptic proteins, yet a direct function in the structural organization of the active zone has not been uncovered in part due to the expression of multiple alternatively spliced isoforms. We recently identified Piccolino, a Piccolo splice variant specifically expressed in sensory ribbon synapses of the eye and ear. Here we down regulated Piccolino in vivo via an adeno-associated virus-based RNA interference approach and explored the impact on the presynaptic structure of mouse photoreceptor ribbon synapses. Detailed immunocytochemical light and electron microscopical analysis of Piccolino knockdown in photoreceptors revealed a hitherto undescribed photoreceptor ribbon synaptic phenotype with striking morphological changes of synaptic ribbon ultrastructure.
    Full-text · Article · Sep 2014 · Frontiers in Cellular Neuroscience
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    • "Splice site prediction analysis using different web tools demonstrated that the site that resulted in the skipping of 101 bp had a very high score as a cryptic splicing donor. The 101 bp-skipped band has not been previously reported as a splicing variant of PCLO in previous studies [19]. Thus, in a minigene assay, activation of the site as a splicing donor resulted in skipping of the last 101 bp of exon 24. "
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    ABSTRACT: The single nucleotide polymorphism (SNP) rs13438494 in intron 24 of PCLO was significantly associated with bipolar disorder in a meta-analysis of genome-wide association studies. In this study, we performed functional minigene analysis and bioinformatics prediction of splicing regulatory sequences to characterize the deep intronic SNP rs13438494. We constructed minigenes with A and C alleles containing exon 24, intron 24, and exon 25 of PCLO to assess the genetic effect of rs13438494 on splicing. We found that the C allele of rs13438494 reduces the splicing efficiency of the PCLO minigene. In addition, prediction analysis of enhancer/silencer motifs using the Human Splice Finder web tool indicated that rs13438494 induces the abrogation or creation of such binding sites. Our results indicate that rs13438494 alters splicing efficiency by creating or disrupting a splicing motif, which functions by binding of splicing regulatory proteins, and may ultimately result in bipolar disorder in affected people.
    Full-text · Article · Oct 2013 · PLoS ONE
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