5’ half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos [small RNA-Seq, ssDRIP-seq]

5’tRFls are small tRNA fragments derived from 5’ half of mature tRNAs. However, it is unknown whether 5’tRFls could feed back to regulate tRNA biogenesis. Here we show that 5’tRFlGly/GCC and 5’tRFlGlu/CTC function to promote transcription of corresponding tRNA genes and are essential for vertebrate early embryogenesis. During zebrafish embryogenesis, dynamics of 5’tRFlGly/GCC and 5’tRFlGlu/CTC levels correlates with that of tRNAGly/GCC and tRNAGlu/CTC levels. Morpholino-mediated knockdown of 5’tRFlGly/GCC or 5’tRFlGlu/CTC down-regulates tRNAGly/GCC or tRNAGlu/CTC levels respectively, and causes embryonic lethality that is efficiently rescued by co-injection of properly refolded corresponding tRNA. In zebrafish embryos, tRNA:DNA and 5’tRFl:DNA hybrids commonly exist on the template strand of tRNA genes. Mechanistically, unstable 5’tRFl:DNA hybrid may prevent the formation of transcriptionally inhibitory stable tRNA:DNA hybrids on the same tRNA loci so as to facilitate tRNA genes transcription. The uncovered mechanism may be implicated in other physiological and pathological processes. For small RNA sequencing, total RNA were extracted respectively from wildtype zebrafish embryos (Tübingen Strain) of 6 chosen stages, and then subjected to small RNA library preparation individually. To investigate the distribution and stability of R-loop on genome, we performed ssDRIP-seq with S9.6 antibody, which specifically recognize RNA:DNA hybrid. Two replicates from wildtype embryos of 256c, sphere and shield stage were collected to perform ssDRIPseq. RNaseH pre-treated genome DNA was used as negative control, which was also performed the standard ssDRIP-seq procedure. Genomic DNA extracted from these samples were fragmentated by restriction enzymes, and then immunoprecipitated by S9.6 antibody.The ssDNA strands in RNA:DNA hybrids were purified and subjected to library preparation.