Fig. 8
Effect of 5′tRFl knockdown on the formation of RNA:DNA hybrids on tRNA loci in zebrafish embryos.
(A) Experimental procedures. MO and cMO doses: 10 ng per embryo. (B) Increased RNA-bound DNA template strand levels on tRNAGlu/CTC genes in 5′tRFlGlu/CTC morphants. ssDNAs isolated from RNA:DNA hybrids in shield stage embryos were used as templates to amplify indicated tRNAGlu/CTC loci. (C and E) Decrease in tRNA and 5′tRFl levels in morphants. Total RNA extracted by neutral phenol:chloroform was used as template for RT-qPCR. (D and F) Increase in DNA-bound tRNA levels accompanied with decrease in DNA-bound 5′tRFls in morphants. RNA isolated from RNA:DNA hybrids was used as template for RT-qPCR. In (B), amplification signal of each tRNA locus was normalized to that in 5% genomic DNA input. In (C) to (F), all qRT-PCR signals were normalized to the level of Chr1-1 tRNAGlu/CTC locus from DNA input. Data are shown as means ± SD. Student’s t test (two-tailed). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Stages of analyzed embryos: Shield stage in (C) and (D) and sphere stage in (E) and (F). |