Fig. 4

Sa1299 is an essential splicing mutant of the smarcad1a gene in zebrafish. (A) Schematic illustration of smarcad1a transcription and T to G single nucleotide mutation in the 5′ end essential spicing site of intron 9. The transcription information is based on the GRCz10 in Ensembl. (B) Normal and aberrant splicing are illustrated in the top row. Proximal cryptic splicing resulted in 13 bps from intron 9 retained in the mRNA. T > G mutation is highlighted blue, and cryptic GT is highlighted with red letters. This result was confirmed by Sanger sequencing (bottom row). (C) Predicted functional domains from full-length protein and truncated proteins (without the functional domains) resulted from premature stop codon (asterisk) due to the proximal cryptic splicing. Amino acid numbers are indicated in the diagram. (D) The mRNA level of ENSDART00000091409 is decreased by RT-PCR in 1dpf sa1299 homozygous embryos, most likely caused by nonsense-mediated mRNA decay. (E) Decrease of smarcad1a ENSDART00000091409 mRNA also is detected by whole-mount in situ hybridization in 1–3 dpf zebrafish embryos. (F) The smarcad1b gene is not affected and still expressed in similar expression domains with smarcad1a in sa1299 homozygotes. The sense probe controls are included in the first two vertical panels