Abstract
Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.
Highlights
Transfer RNAs are essential molecules required for decoding messenger RNAs into proteins
Detecting a G34 on a sequencing read that unequivocally mapped to an ANN Transfer RNAs (tRNAs), strongly supports that the A34 has been deaminated into Inosine at position 34 (I34)
RNA sequencing (RNAseq) is a powerful tool to detect and quantify RNA species. The use of this technology to detect and quantify tRNAs has been hampered due to the difficulties in sequencing these type of RNAs [32,33,34]. It has only been in the past few years that RNAseq protocols and bioinformatics analyses have been optimized for studying tRNAs and even so, until now, such strategies proved useful to answer only a particular set of biological questions
Summary
Transfer RNAs (tRNAs) are essential molecules required for decoding messenger RNAs (mRNAs) into proteins. They contain a nucleotide triplet (anticodon) composed of residues 34, 35 and 36 of the tRNA molecule that recognizes nucleotide triplets (codons) on mRNAs. tRNAs need to be heavily modified post-transcriptionally in order to become fully active. Modifications at the anticodon arm are usually seen as enhancers of the efficiency and fidelity of mRNA decoding [1]. The residue at position 34 of the tRNA (the first nucleotide of the anticodon) is frequently modified to allow the base 34 to ‘wobble’ and pair with non-canonical bases, allowing certain tRNAs to recognize more than one mRNA codon [2]
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