Fig. 5

(A) Injection of 5′tRFl^Glu/CTC or 5′tRFl^Gly/GCC-mimetics leads to no obvious defects till 36 hpf. Scale bar, 100 μm. (B and C) 5′tRFl^Gly/GCC and 5′tRFl^Glu/CTC positively regulate related tRNA levels. Embryos at the 1c stage were injected with 0.6 pmol of RNA oligo and collected at the 256c stage for RT-qPCR (B) or Northern blot (C) as described in Fig. 4 (B and C). (D) Illustration of in vitro tRNA gene transcription system (ivTGTS). The upstream sequences (red line) and transcription sequences (green box) of three tRNA^Glu/CTC loci were used to construct reporter plasmids by each fusing to the artificial reporter sequence F30-Broccoli (F30-Br). TSS, transcription start site; TTS, transcription termination site. (E) Effectiveness test of F30-Broccoli reporter transcription in ivTGTS by RT-PCR. Presynthesized GFP mRNA was added to serve as an exogenous reference. (F and G) Effect of 5′tRFl^Glu/CTC-mimetic (F) or 5′tRFl^Glu/CTC-MO (G) on F30-Broccoli reporter transcription in ivTGTS. Doses (per reaction): 1 μg of DNA mix; MO, 1 μg; RNA oligos, 60 pmol. For all experiments in this figure, data are shown in average with ±SD from three independent experiments. ns, nonsignificant with P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Student’s t test, two-tailed).