Neuron-specific repression of alternative splicing by the conserved CELF protein UNC-75 in Caenorhabditis elegans

TL;DR

This study shows how the protein UNC-75 controls neuron-specific splicing in C. elegans by binding to specific DNA sequences.

Contribution

The study identifies conserved UNC-75 binding motifs that repress alternative splicing in neurons.

Findings

UNC-75 loss or cis-element mutations cause de-repression of a neural-repressed exon in C. elegans.

Mis-expression of UNC-75 in muscle cells induces neuron-like exon skipping.

UNC-75 motifs in splice sites increase exon skipping in unrelated splicing events.

Abstract

Tissue-regulated alternative exons are dictated by the interplay between cis-elements and trans-regulatory factors such as RNA-binding proteins (RBPs). Despite extensive research on splicing regulation, the full repertoire of these cis and trans features and their evolutionary dynamics across species are yet to be fully characterized. Members of the CUG-binding protein and ETR-like family (CELF) of RBPs are known to play a key role in the regulation of tissue-biased splicing patterns, and when mutated, these proteins have been implicated in a number of neurological and muscular disorders. In this study, we sought to characterize specific mechanisms that drive tissue-specific splicing in vivo of a model switch-like exon regulated by the neuronal-enriched CELF ortholog in Caenorhabditis elegans, UNC-75. Using sequence alignments, we identified deeply conserved intronic UNC-75 binding motifs overlapping the 5′ splice site and upstream of the 3′ splice site, flanking a strongly neural-repressed alternative exon in the Zonula Occludens gene zoo-1. We confirmed that loss of UNC-75 or mutations in either of these cis-elements lead to substantial de-repression of the alternative exon in neurons. Moreover, mis-expression of UNC-75 in muscle cells is sufficient to induce the neuron-like robust skipping of this alternative exon. Lastly, we demonstrate that overlapping an UNC-75 motif within a heterologous 5′ splice site leads to increased skipping of the adjacent alternative exon in an unrelated splicing event. Together, we have demonstrated that a specific configuration and combination of cis elements bound by this important family of RBPs can achieve robust splicing outcomes in vivo.

Pilaka-Akella et al. are still learning the code used by the cell to create tissue-specific splicing patterns. Here, using C. elegans, the authors identify UNC-75, a highly conserved RNA binding protein from the CELF family, as a potent repressor of a model tissue-biased exon. UNC-75 regulates exon skipping in neurons by binding to multiple motifs in the introns flanking the exon, and mis-expression of UNC-75 in muscle cells potently represses exon inclusion. Thus, their work sheds further light on RNA processing mechanisms.

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