Fig. S7

The prp19 morpholino inhibits proper splicing of the prp19 pre-mRNA transcript. (A) Schematic of the zebrafish prp19 gene with exons shown as boxes and introns shown as lines between boxes. The e3i3 morpholino targets the splice donor site at the border of exon 3 and intron 3. In (B), F1 and R2 primer-based PCR will amplify from exon 2 to intron 3. Properly spliced mRNA should not give rise to PCR product. Inclusion of intron 3 should give rise to a 245-basepair product. (B) RNA from embryos injected with mismatch morpholino controls or prp19 e3i3 morpholino was harvested at 24 hpf, reverse transcribed with oligo-dT primers, and analyzed by PCR using primers shown in (A). Bands were excised from the gel, cloned into pGEM-T-Easy, and sequenced. Lower-case letters point to bands on the gel which correspond to regions of the prp19 gene shown below. When embryos were injected with the e3i3 morpholino, F1/R2 primers gave rise to a band representing inclusion of intron 3 (b). This should cause premature termination the Prp19 protein since a stop codon is present in the middle of the intron. Unexpectedly, all samples gave rise to a 330-basepair band (a) which is likely derived from the presence of contaminating genomic DNA but does not change interpretation of the data.