removed by ricin (430). Abasic sites are also found in tRNAs where certain bases are labile, such as wybutosine (Y or yW) at position 37 in tRNA<sup>Phe</sup> (431), as well as the modified base m<sup>7</sup>G at position 46 in other tRNAs (432). Interestingly, removal of yW<sub>37</sub> significantly impairs phenylalanine acceptance (433). It can be expected that in the near future new tools will be available. The recent characterization of methylpurine DNA glycosylase, which generates abasic RNAs on RNA-DNA hybrids (434), may become programmable in the future, opening up exciting prospects in the search for tRNA identity elements.

## Future directions for tRNA identity research

Over the past two decades, the field of tRNAs has entered a new era in which the prototypical concept of tRNA identity has been challenged and progressively refined in response to new discoveries. The increasing importance of auxiliary protein factors alongside aaRSs in tRNA biology is significant.

This has produced a paradigm shift, with semantic changes illustrated by the concepts of ‘quality control’ underlying tRNA aminoacylation (361) and of ‘code biology’ (435) extending the ‘operational RNA code’ (10). More generally, tRNA is a pivotal marker in evolution, illustrated by breakthroughs in eukaryotic tRNA biology (2,3). On the other hand, large-scale searches of tRNA sequences by bioinformatics methods revealed patterns of importance of tRNA positions related to identities and suggested that ‘almost nothing in most tRNA positions is even close to random’ (385). Therefore, the long-term prospect will be to decipher globally in given tRNAs the functional role of nucleotides at these positions.

What are the most immediate prospects? Only a few aminoacylation identities are known in organelles, and the field is completely open for apicoplast and chloroplast tRNAs. Furthermore, it is crucial to better understand identities outside of aminoacylation, especially for tRNA maturation and splicing. To do this, known and predicted determinants must be deconvoluted in terms of aminoacylation and associated functions. In addition, much remains to be discovered in tRNA biology, including its dynamic regulation under specific environmental and ecological conditions. In particular, how the cellular tRNA pool varies in response to various metabolic contexts is poorly understood and needs to be quantified. Epistatic effects that cover combined effects of mild mutations in tRNAs and/or aaRSs are other challenges (387,436). To achieve these ambitious goals, appropriate biochemical, genetic, computational and deep sequencing tools are needed. Finally, it is becoming clear that the identity of tRNAs recapitulates the evolutionary history of protein synthesis and life, and this puzzle must be clarified to understand the origins of tRNAs.

## DATA AVAILABILITY

All data are available from the authors upon request.

## SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

## ACKNOWLEDGEMENTS

We would like to thank all our colleagues, both local, national, and worldwide, with whom we have had many discussions, exchanges of ideas, and collaborations in the field of tRNA over the years. Their names and contributions are listed in the references.

## FUNDING

This work was supported by the French ‘Centre National de la Recherche Scientifique’; ‘University of Strasbourg’, and ‘Agence Nationale de la Recherche’ [ANR-17-CE11-0024]. *Conflict of interest statement.* None declared.

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APPENDIX

Definitions

Catalytic efficiency: defined by the *k*<sub>cat</sub>/*K*<sub>M</sub> ratio of aminoacylation reactions, with *k*<sub>cat</sub> the catalytic rate constant and *K*<sub>M</sub> the Michaelis constant representing an approximation of the inverse of tRNA affinity for an aaRS, so that ‘L’ = (*k*<sub>cat</sub>/*K*<sub>M</sub>)<sub>native</sub>/*(*k*<sub>cat</sub>/*K*<sub>M</sub>)<sub>mutant</sub> (with ‘L’ representing the loss of catalytic efficiency).

Classes of tRNAs: based on the presence of a small variable region of 4/5 nt (class I) or a large variable region with an extra loop (class II).
**Classes and subclasses of aaRSs:** defined based on the structure and peptide motifs present in their catalytic sites.

**Identity determinant:** nucleoside or chemical groups on a nucleoside, as well as structural elements (mainly in tRNAs) essential for biological activity (in proteins, this concerns amino acids).

**Identity set:** a set of key determinants for specific activities.

**Identity rules:** account for the operational functioning of identity determinants in taxonomic groups (originally they applied exclusively to the aminoacylation reaction of tRNAs and were considered ‘universal’ if they were conserved in the three domains of life).

**Isoacceptor tRNAs:** tRNAs that accept the same amino acid and may have different anticodons due to genetic code degeneracy.

**Isodecoder tRNAs:** tRNAs with the same anticodon sequence that decode the same codon. They belong to the same class of isoacceptors but have sequence differences elsewhere in the tRNA body.

**Strength of aminoacylation identity determinants:** measured *in vitro* by losses of catalytic efficiency of aminoacylation reactions of tRNA upon mutation at identity positions. Alternatively, strength can be measured *in vivo* by *E. coli* suppressor genetics that utilizes a reporter dihydrofo late reductase gene with the amber mutational position 10. **Tertiary interactions in tRNA:** interactions that stabilize the structure of tRNA. They are mainly located in the core of the tRNA molecule and include, for example, interactions between the D stem and variable loop, T loop and anti- codon loop, and $U_8$ with $A_{14}$.

**tRNA domains:** D stem/loops from $N_{10}$ to $N_{25}$ with loops from $A_{14}$ to $A_{21}$ divided into α and β subdomains (sizes from 2 to 4 nts) located down- and upstream of the constant $G_{18}$ and $G_{19}$); anticodon stem/loops from $N_{27}$ to $N_{43}$ with 7 nt loops ($N_{32}$–$N_{38}$); variable regions (VR) from $N_{44}$ to $Y_{48}$ with smallVRs (4 or 5 nt) or largeVRs (also named extra arm) (up to 16 nt from positions 47a to 47p); T stem/loops from $N_{47}$ to $N_{65}$ with 7 nt loops ($T_{54}$–$N_{60}$); connecting Ns: $U_8N_9$ and $N_{26}$.