mec-8 and exc-7 combines to regulate splicing of unc-52 exons.
(A) mec-8 and exc-7 show antagonistic regulation of exons 17–18 in unc-52 at the L1 stage. (B) Reduction of activity of both mec-8 and exc-7 show additive effects on unc-52 splicing patterns at the L1 stage. (C) Study of unc-52 splicing patterns in vivo using a bichromatic splicing reporter. The bichromatic splicing reporter was constructed using the same method as described by Norris et al. . Skipping of exons leads to expression of GFP, while inclusion of exon(s) results in a frameshift that leads to readthrough of the GFP reading frame, and resulting expression of mCherry. Worms were subjected to RNAi at the L4 stage, and late L1/L2 worms were imaged. A dsRNA that targets a C. briggsae gene was used as a non-targeting control. Transgenic mec-8 mutant worms show increased inclusion of the unc-52 minigene exons in hypodermal cells, while worms treated with exc-7 RNAi show increased exon skipping. Transgenic mec-8 worms treated with exc-7 RNAi show both exon-included and exon-skipped isoforms in hypodermal cells. Examples of hypodermal cells that express both isoforms are highlighted with white arrowheads.