Fig. 4: Detection of novel alternative first exon in sax-3.

A Structure of the gene sax-3/ROBO indicating the novel exon 5.5 and alternative 5’ splice site at the end of exon 9 (green), and the annotated alternative 3’ splice site at the start of exon 11 (red). Bottom: Enlarged 3’ end of the locus. B Local quantification of LSVs in the representative neurons AVL and AVM for the novel alternative first exon 5.5 (left), the novel alternative splice site (middle), and the annotated alternative splice site (right). Violin plots represent the posterior distribution computed by MAJIQ. C, D Validation by RT-PCR of the novel exons in cDNA from whole animals (N2 wild type). Alternative first exon 5.5 (C), top: Location of primers relative to the exon. Left, the primer pair only amplifies the annotated transcript including exons 1-5; right, the primer pair only amplifies the novel transcript using exon 5.5 as first exon. Arrowheads: the expected product sizes (307 bp, 286 bp). Alternative 5’ splice site in exon 9 (D), top: Location of the primers relative to the event. Left, the primer pair only amplifies the novel transcript with shorter exon 9; right, the primer pair only amplifies the annotated transcript with longer exon 9. Arrowheads: expected product sizes (242, 248 bp). Three biological replicates along with a control without reverse transcriptase. Molecular weights reported in base-pairs. Source data are provided as a Source Data file. E Protein structure of SAX-3, indicating the impact of the novel splice variants. Top: Overall structure of annotated isoform SAX-3A. Bottom: Overall structure of the novel short isoform starting at novel exon 5.5 (SAX-3S). The position of the novel splice site in exon 9 is indicated. F Structure of the orthologs of sax-3 in C. briggsae (Cbr-sax-3) and C. brenneri (Cbn-sax-3). Top: Gene models. Bottom: RNA-Seq data. Red arrowheads denote the orthologs of exon 5.5. G Protein sequence alignment of the short isoforms of SAX-3 orthologs from C. elegans, C. briggsae, and C. brenneri.