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. Overall design: 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.