Mammals have nine different homologues (ALKBH1–9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm⁵U) from its precursor 5-carboxymethyluridine (cm⁵U). In this study, we report that (R)- and (S)-5-methoxycarbonylhydroxymethyluridine (mchm⁵U), hydroxylated forms of mcm⁵U, are present in mammalian , and , respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm⁵U are generated from mcm⁵U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm⁵U into (S)-mchm⁵U in . These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon–anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.

To date the only tRNAs containing nucleosides modified with a selenium (5‐carboxymethylaminomethyl‐2‐selenouridine and 5‐methylaminomethyl‐2‐selenouridine) have been found in bacteria. By using tRNA anticodon‐stem‐loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2‐selenouridine synthase (SelU) converts S2U‐RNA to Se2U‐RNA in a two‐step process involving S2U‐RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U‐RNA (with SePO₃³⁻). No ‘direct’ S2U‐RNA→Se2U‐RNA replacement is observed in the presence of SelU/SePO₃³⁻ only (without ppGe). These results suggest that the in vivo S2U→Se2U and S2U→geS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events – geranylation (S2U→geS2U) and selenation (geS2U→Se2U).

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.