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. 2014 Oct 10;42(20):12768–12778. doi: 10.1093/nar/gku941

Figure 5.

Figure 5.

Viability and motoric function of zebrafish embryos injected with CUG repeat RNA is modified by pseudouridylation. (A) DMPK and GFP control constructs injected into zebrafish. These RNAs were transcribed in vitro with either all UTP or all Ψ-NTP. RNA containing the DMPK 3′ UTR has 91 CUG repeats. (B) Viability of zebrafish embryos at 24 h post fertilization after injection with in vitro-transcribed GFP or GFP:(CUG)91 RNA containing either uridine or Ψ. There was no significant difference in the viability of embryos injected with GFP with and without Ψ. Embryos injected with (CUG)91 RNA transcribed in vitro with 100% Ψ exhibit significantly enhanced viability compared to uridine-containing (CUG)91 RNA. N>400/>500 zebrafish per genotype over at least three independent experiments. For GFP versus (CUG)91 the P value was 0.014. For RNA (Ψ(CUG)91 versus (CUG)91 the P value was 0.030. P < 0.05 by Fishers exact test. (C) Spontaneous coiling movements in 24 hpf zebrafish embryos injected with the indicated in vitro-transcribed RNAs. Coiling data is graphed as the number of spontaneous coiling movements/minute. (CUG)91 RNA-injected embryos exhibit impaired early muscle development and function, with decreased spontaneous coiling at 24 hpf. In contrast, embryos injected with 100% Ψ (CUG)91 RNA exhibit normal spontaneous coiling rates compared to GFP-injected embryos (P < 0.0001). N>100/ zebrafish per genotype over three independent experiments were analyzed. All groups were significantly different by a Kruskal Wallis ANOVA. * indicates P < 0.0001 by Post-hoc Mann-Whitney U test for viability. * indicates P < 0.05 and **** indicates P < 0.0001 by standard unpaired, two-tailed t test for spontaneous coiling.