Figure 5.

In Vivo Data: Individuals’ RNA and Zebrafish

(A) Deregulation of PUF60-dependent alternative splicing in affected individuals. Quantitation of RT-PCR analysis of alternatively spliced mRNA isoforms from SMN2, PUF60, BIN1, and APP from relatives (n = 6) or proband (n = 4) with a mutation or deletion of PUF60.

(B) Quantification of the total body length was performed in embryo batches injected with puf60a MO alone or puf60a MO along with either human wild-type (WT) or mutant PUF60 (p.His169Tyr) messages. The p.His169Tyr mutation was introduced in the long and short isoforms. Bars represent the average length of 80 embryos at 3 dpf, which were scored blind to injection cocktail. Injection of long WT and mutant mRNAs rescued the body length phenotype observed in puf60a morphants (p < 0.0001; two-tailed t test comparisons). The short mutant PUF60 mRNA failed to rescue the phenotype and showed no significant difference with the embryos injected with the puf60a MO alone. Data are shown as the mean ± SEM (three independent experiments). Hatch sign (#) indicates nonsignificant.