Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria-which models tuberculous granulomas-are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

5-Taurinomethyluridine (τm5U) and 5-taurinomethyl-2-thiouridine (τm5s2U) are located at the wobble position of human mitochondrial (hmt) tRNALeu(UUR) and tRNALys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNALeu(UUR) and hmt-tRNALys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm5U and τm5s2U into 17-mers related to the sequence of the anticodon arms hmt-tRNALeu(UUR) and hmt-tRNALys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm5s2U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.

5-Carboxymethylaminomethyluridine (cmnm5U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) are located at the wobble position in several cytosolic and mitochondrial tRNA sequences. In this paper, we report the first site-selected incorporation of cmnm5U and cmnm5s2U into RNA sequences by phosphoramidite chemistry on a CPG solid support. Trifluoroacetyl and 2-(trimethylsilyl)ethyl were selected for the protection of the amine and carboxyl functions, respectively.

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2',3'-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)3) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm(5)(s(2))U) and 5-taurinomethyl(-2-thio)uridine (τm(5)(s(2))U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2',3'-O-isopropylidene-2-thiouridine is also reported.

Tautomeric shifts of five-modified uridines have been established in, Escherichia coli tRNAVal cmo5UAC, as well as in the human cytoplasmic tRNALys3 mcm5s2UUU. These shifts enable the recognition of multiple codons with the wobble base pair in Watson–Crick geometry, suggesting an energetic preference for perhaps an enhanced efficiency with this conformation. Thus, modifications at the fifth-position of wobble position uridines appear to be a common mechanism for expanding tRNA’s codon recognition. One deviation from the universal code which is particularly interesting is the reading of AUG and the universal isoleucine codon AUA as methionine in both the A- and P-sites of the mitochondrial ribosome of most metazoans. We have discovered a mechanism by which five-formylcytidine (f5C) at the wobble position of human mitochondrial tRNAMet (hmtRNAMet f5CAU) expands the decoding ability of tRNA, enabling it to read AUA as methionine. We have also found that another modified cytidine can restrict wobble-base formation. This underscores the ability of cytidines, as well as uridine, modifications within the anticodon loop to modulate the decoding ability of the tRNA, providing an insight into decoding mechanisms and evolution of the genetic code.

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 μM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons.

We present a reproducible protocol for the site-specific incorporation of 5-methylaminomethyl-2-thiouridine (mnm5s2U) into a model RNA fragment and, together with 2-methyladenosine (m2A), into the native sequence of the Escherichia coli tRNAGlu2 anticodon arm (E. coli ASLGlu2). This approach is also utilized for the synthesis of oligomers modified with multiple 2-thiouridines.

Incorporation of 2-thiocytidine (s2C) into a DY647 labelled 17-mer RNA sequence corresponding to the anticodon stem loop of Staphylococcus aureus tRNAArg has been achieved by phosphoramidite chemistry. Key to the success was the use of dilute iodine for the P(III) to P(V) oxidation step plus the choice of the trimethylsilylethyl (tse) ester as protection for the threonyl carboxyl group of t6A. In trial experiments with N4-benzoyl-2-thiocytidine, the use of dilute t-BuOOH in toluene as an alternative oxidising agent caused substantial desulfurization.

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA(Lys3)(UUU) as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA(Lys3)(UUU), the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL(Lys3)) were studied. NCp7 bound the hASL(Lys3)(UUU) modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(37)) with a considerably higher affinity than the unmodified hASL(Lys3)(UUU) (K(d)=0.28±0.03 and 2.30±0.62 μM, respectively). NCp7 denatured the structure of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) more effectively than that of the unmodified hASL(Lys3)(UUU). Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K(d)=0.55±0.10 μM) and specificity for the ASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 μM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA(Lys3)(UUU) have been found to be important determinants of NCp7's recognition prior to the tRNA(Lys3)(UUU) being annealed to the viral genome as the primer of reverse transcription.

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. Uridines at the wobble position of transfer RNA anticodons are usually modified to allow efficient decoding of messenger RNA codons. In this study, ALKBH8 is shown to be a bifunctional transfer RNA modification enzyme required for the formation of a novel diastereomeric pair of modified wobble uridines.

tRNA is the most heavily modified of all RNA types, with typically 10–20% of the residues being post‐transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar. Furthermore, the tRNA has to be investigated with single‐nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNALys(UUU) from Trypanosoma brucei, and provide a complete overview of its post‐transcriptional modifications. The first step was MALDI‐TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass‐changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher‐order tandem MS. Phylogenetic comparison with modifications in tRNALys from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNALys(UUU), where U47 exhibited a novel modification, 3‐(3‐amino‐3‐carboxypropyl)‐5,6‐dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2‐methylthio‐N6‐threonylcarbamoyl‐modification, and a major fraction with A37 being modified by a 294.0‐Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (Ψ(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNALys3, to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3′-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5′-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNALys3. The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s2U34, and pseudouridine, Ψ39, appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U162•Ψ39 and G163•A38, that maintained a reasonable A-form helix diameter. The tRNA's s2U34 stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Ψ39 stabilized the adjacent mismatched pairs.

Replication of HIV requires base pairing of the reverse transcriptase primer, human tRNALys3, to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence and the tRNA's 3′‐terminus, an important discriminatory, secondary contact occurs between the viral A‐rich Loop I and the modified, U‐rich anticodon domain of tRNALys3. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral serosubtypes G and B were analyzed by UV and CD and the structure of one duplex was determined using NMR spectroscopy and restrained molecular dynamics. Thiouridine, s2U34, and pseudouridine, ?39, modifications appreciably stabilized the interaction of the anticodon region with the viral RNAs. The structure of the duplex composed of the HIV‐1 Loop I bound to a doubly modified tRNA's anticodon region resulted in two coaxially stacked A‐form RNA stems separated by two mismatched base pairs, U?? and G?A, that maintained a reasonable helix diameter. Thus, the important interaction of the primer's anticodon region with the viral RNA is strengthened by s2U34 and ?39 modifications that facilitate duplex interaction. This research was supported by a National Institutes of Health grant and National Science Foundation grants.