Fig. 9
tRNA gene transcription is regulated by tRNAs and 5′tRFls.
(A to C) RNASEH1 overexpression elevates endogenous tRNA levels. Wild-type (WT) (A and C) or Tg(Chr.21-1:tRNAGlu/CTC-F30-Broccoli) (B) embryos were injected with 300 pg of NLS-hRNASEH1-GFP [composition shown on top of (A)] or GFP mRNA at the 1c stage and immunostained for GFP observation at indicated stages (A) or at the 256c stage for examining expression levels of F30-Broccoli (B) or several tRNAs by RT-qPCR (C), which were normalized to 18S rRNA levels. Note that NLS-hRNASEH1-GFP was enriched in nuclei at 4 hpf. (D to F) Length-dependent regulation of tRNA gene transcription. Three tRNAGlu/CTC oligos with different lengths (D) were synthesized, and they have different minimum free energy (ΔG37°C) and melting temperature (Tm) for DNA:RNA hybrids (E). 1c stage Tg(Chr.21-1:tRNAGlu/CTC-F30-Broccoli) transgenic embryos were injected with individual RNA oligos at 60 fmol per embryo and harvested at the 256c stage for examining F30-Broccoli expression level by RT-qPCR analysis (F). The reporter expression level was normalized to 18S rRNA level. For data in (B), (C), and (F), the average with ±SD from three independent experiments is given. ns, nonsignificant with P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test, two-tailed). (G) Working model. Left: Scenario of tRNA gene (tDNA) transcription in wild-type zebrafish embryos. The major wave of zygotic gene activation for protein-coding genes occurs at the 1kc stage. Before the sphere stage, tDNA transcription may be slow because of tRNA association with the template strand. After the sphere stage, increasing amount of 5′tRFls, possibly with the aid of other factors, takes over the tRNA for binding with the template strand. Right: Scenario of tDNA transcription in embryos depleted of 5′tRFl in which full-length tRNA forms stable tRNA:DNA hybrids on the template strand and thus hinders tDNA transcription. DAPI, 4′,6-diamidino-2-phenylindole. |