Fig. S6

Test of the a2aa splice-blocking morpholino by RT-PCR and sequencing of truncated transcript, related to Figure 5.

(A) RT-PCR showing reduced amount of spliced a2aa mRNA in larvae injected with 5 or 10 ng a2aa MO at 4, 5, and 6 dpf. 10 larvae from each condition and stage were used for cDNA synthesis. The a2aa morpholino blocks the splice donor site of the first coding exon (dashed underlined in red) leading to a decreased amount of PCR product as the primers anneal to the first and second exons (underlined sequence). A truncated transcript appeared in the presence of the MO. Equal amounts of cDNA template are indicated by amplification of elongation factor 1-α, using the primers (5′-tcaccctgggagtgaaacagc-3′) and (5′-acttgcaggcgatgtgagcag-3′). (B) Sequence of the normal a2aa cDNA and splice junction (black /). The truncated transcript formed in the presence of the splice blocking MO used an alternative splice junction (red /), leading to the deletion of the sequence labeled in red. This deletion causes a frameshift that creates a premature stop codon (double underlined), thereby leading to a truncated protein of 149 amino acids that only includes 55 out of the 442 amino acids of the full-length A2aa protein.