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Fig. S2 Nonsense mediated decay and nonsense mediated alternative splicing of etfdh transcript in xav, and morpholino knock down of etfdh. A. qRT-PCR showed that there is a significant, ∼80% reduction of etfdh mRNA in xav, likely due to nonsense mediated decay. N = 3 pools of 20 embryos each for WT and xav; Student′s t test, * p<0.0001. B. xav mutants showed nonsense mediated alternative splicing. As a result of the mutation, which resides in exon11, the exon10-exon13 junctions are mis-spliced in mutants, resulting in transcripts that are predicted to encode proteins lacking critical domains or truncated. Blue arrows indicate primer location. These results suggest that the xav mutation is likely to be loss of function. C. A splice-blocking morpholino against intron2-exon3 (MOI2E3) was designed for etfdh. D. Injection of 8 ng etfdh MOI2E3 in WT embryos, results in >80% reduction of the normal transcript at 2 and 3 dpf, producing a mis-spliced transcript that lacks exon3. Red arrow indicates the MO location and blue arrows indicate primer location.