Detection of posttranscriptional inosine modification at the precursor tRNA level

Transfer RNAs (tRNAs) are key adaptor molecules in the protein translation machinery. In order to become fully active, they are heavily modified posttranscriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalysed by the heterodimeric Adenosine Deaminase Acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing, we show that this modification takes place in human tRNAs at the precursor tRNA level and as this precursor maturates. We also functionally validated the human genes encoding for hetADAT and show that in cells, HsADAT2 and HsADAT3 co-localize in nucleus. Lastly, we knocked down HsADAT2 and show for the first time that it is possible to modulate the levels of I34 modification on all human hetADAT tRNA substrates. These findings give mechanistic insights on the biology of tRNA modifications and the tools reported in this study will prove useful in the future to address the exact molecular role of I34 modification on protein translation.