Date of Award
Doctor of Philosophy (PhD)
The participation of 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 read mapping efforts. To address these issues, I developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, I further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. My results show that approximately half of all predicted tRNA genes are transcribed in human cells, suggesting that the tRNA genomic space is more contracted than previously thought as a result of regulation of expression. I also report predominant nucleotide modification sites, their order of incorporation, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies I present a human tRNA reference set, and determine expression levels of mature and processing intermediates of tRNAs in human cells. The technical advances provided by hydro-tRNAseq are applied towards the molecular diagnosis of a genetic neurodevelopmental syndrome, caused by mutations in the tRNA processing factor CLP1. Finally, I harness this novel experimental and computational expertise towards the identification of the endonuclease complex C3PO as a novel processing factor of human tRNAs. I carry out a transcriptome-wide analysis of C3PO targets, identify its binding sites and motifs, and provide insights into its biochemical and biological functions.
Gogakos, Tasos, "Characterizing Human Transfer RNAS by Hydro-TRNASEQ and PAR-CLIP" (2018). Student Theses and Dissertations. 416.