Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP

Abstract

Summary 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 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, their order of introduction, and 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.

Translational efficiency across healthy and tumor tissues is proliferation‐related

“…To validate the use of small RNA‐seq for tRNA quantification, we first compare tRNA levels determined in HEK293 by well‐established tRNA sequencing methods (Hydro‐tRNAseq and demethylase‐tRNAseq) (Zheng et al , 2015a; Gogakos et al , 2017a; Mattijssen et al , 2017a), with those obtained by small RNA‐seq. Then, we quantify the tRNA repertoire of five cell lines using Hydro‐tRNAseq and perform small RNA‐seq in parallel.…”

“…In order to understand such changes in codon-anticodon coadaptation, orthogonal datasets of gene expression including tRNA quantification are required, which needs to overcome the challenges of strong secondary structures and abundant chemical modifications. Recent technological developments have paved the way for sensitive high-throughput tRNA sequencing across tissues and conditions (Zheng et al, 2015a;Gogakos et al, 2017a). Aside from these methods and despite the lower coverage, tRNA reads can also be detected from generic small RNA-seq datasets (Guo et al, 2015(Guo et al, , 2016Pundhir & Gorodkin, 2015;Torres et al, 2015a;Hoffmann et al, 2018).…”

The Conserved RNA Exonuclease Rexo5 Is Required for 3′ End Maturation of 28S rRNA, 5S rRNA, and snoRNAs

“…At the latest viable time point, size and tissue morphologies of homozygous mutants were comparable to wild-type L2 larvae. Considering that RNAs in the nucleolus, the site of Rexo5 localization, range in size from less than 100 nucleotides (nt) for snoRNAs to thousands of nucleotide for rRNAs, we applied a small RNA sequencing protocol using partially hydrolyzed total RNA as input RNA (Gogakos et al, 2017). We analyzed each noncoding RNA group separately, first examining small abundant noncoding RNAs (20–200 nt), specifically snoRNAs, snRNAs (grouped into one category snoRNA/snRNA), and tRNAs (Figure 3A,C, S3A).…”

The cellular landscape of mid‐size noncoding RNA

“…For example, snRNA modifications, like base methylations, 2′‐O‐methylation, and pseudouridylation, play an important role in spliceosome assembly and regulate splicing efficiency (Bohnsack & Sloan, ; Kiss, ). tRNAs are the most modified RNAs, often by deamination to inosine, various forms of methylation and acetylation (Gogakos et al, ; Lorenz, Lunse, & Morl, ). Modification of tRNA affects folding, stability and function including the decoding of the wobble position and frameshifting (El Yacoubi, Bailly, & de Crecy‐Lagard, ; Lorenz et al, ; Nachtergaele & He, ).…”