Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP
Tasos Gogakos,
Miguel Brown,
Aitor Garzia,
Cindy Meyer,
Markus Hafner,
Thomas Tuschl
Affiliations
Tasos Gogakos
Howard Hughes Medical Institute and Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
Miguel Brown
Howard Hughes Medical Institute and Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
Aitor Garzia
Howard Hughes Medical Institute and Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
Cindy Meyer
Howard Hughes Medical Institute and Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
Markus Hafner
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Diseases, 50 South Drive, MSC 8024, Bethesda, MD 20892, USA
Thomas Tuschl
Howard Hughes Medical Institute and Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
The participation of tRNAs in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes and curation of tRNA sequences. This has been challenging because RNA secondary structure, nucleotide modifications, and tRNA gene multiplicity complicate sequencing and 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 nucleotide modification sites and their order of introduction, and we identify tRNA leaders, trailers, and introns. 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.
