Inosine modifications in human tRNAs are incorporated at the precursor tRNA level

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.

Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method

“…The major drawback of RNAseq is that it often results in the detection of partially modified precursor tRNAs, rather than fully mature tRNAs 19,27 .…”

“…This is consistent with at least one of the aforementioned modifications inducing a detectable 'mutation' on the PCR amplicon. Of note, m 1 I37 has been reported to induce such a mutation as detected by RNAseq 15,19 . To show the robustness of the method using in vivo tRNA samples, we performed three independent replicates for this experiment ( Figure 7B).…”

“…After ruling out possible contamination of the PCR reaction with genomic DNA ( Figure 6B) we concluded that part of the obtained amplicons were derived from unmodified precursor tRNA species. It is well known that RT of fully matured tRNAs is challenging and that in complex RNA samples, there is a bias towards reverse transcribing precursor tRNAs over mature tRNAs 19,[27][28][29] is only partially cleaved ( Figure 7A). This is consistent with at least one of the aforementioned modifications inducing a detectable 'mutation' on the PCR amplicon.…”

Protein folding and tRNA biology

“…This method presents several biases: ligation of adaptors is not equally efficient on all molecules, modifications cannot be detected, the fact of being a method based on RT-PCR means that just a few molecules can be fully sequenced. Nevertheless, several groups have used RNA sequencing datasets to detect modified tRNA residues, exploiting the fact that not all sequencing errors are technical artifacts, but, conversely, they often conceal biological marks of post-transcriptional RNA modifications sites (Ebhardt et al 2009;Iida et al 2009;Findeiss et al 2011;Ryvkin et al 2013;Torres et al 2015). By analyzing mismatches between sequencing reads and the genomic region where those reads mapped, as well as by analyzing special read patterns, they have been able to distinguish known and potentially novel post-transcriptional base modifications on tRNAs and, in some cases, also allowing a Brelative^quantification of tRNA species (Torres et al 2015).…”

tRNA biology in the omics era: Stress signalling dynamics and cancer progression

“…Based on the widespread differences between the RNA and DNA sequences, standard RNA-seq experiments have been used to identify numerous instances of A-to-I editing [107], and revealed that inosine is incorporated both at the pre-tRNA and mature tRNA level [108]. While this bioinformatics approach is extremely powerful, great care must be taken in the downstream analysis to discriminate true modifications from potential false positives [109].…”