GSE95683
Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP
The participation of transfer RNAs (tRNAs) in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and sequencing read mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report predominant nucleotide modification sites, their order of introduction, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies, we present a human tRNA atlas, and determine expression levels of mature and processing intermediates of tRNAs in human cells. Overall design: Four samples for hydro-tRNAseq in HEK293 cells. Two samples for SBB PAR-CLIP in HEK293 cells.