Grazyna Leszczynska

Nucleoside modifications in the regulation of gene expression: focus on tRNA.

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.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

C5-substituents of uridines and 2-thiouridines present at the wobble position of tRNA determine the formation of their keto-enol or zwitterionic forms - a factor important for accuracy of reading of guanosine at the 3′-end of the mRNA codons

Abstract Modified nucleosides present in the wobble position of the tRNA anticodons regulate protein translation through tuning the reading of mRNA codons. Among 40 of such nucleosides, there are modified uridines containing either a sulfur atom at the C2 position and/or a substituent at the C5 position of the nucleobase ring. It is already evidenced that tRNAs with 2-thiouridines at the wobble position preferentially read NNA codons, while the reading mode of the NNG codons by R5U/R5S2U-containing anticodons is still elusive. For a series of 18 modified uridines and 2-thiouridines, we determined the pKa values and demonstrated that both modifying elements alter the electron density of the uracil ring and modulate the acidity of their N3H proton. In aqueous solutions at physiological pH the 2-thiouridines containing aminoalkyl C5-substituents are ionized in ca. 50%. The results, confirmed also by theoretical calculations, indicate that the preferential binding of the modified units bearing non-ionizable 5-substituents to guanosine in the NNG codons may obey the alternative C-G-like (Watson–Crick) mode, while binding of those bearing aminoalkyl C5-substituents (protonated under physiological conditions) and especially those with a sulfur atom at the C2 position, adopt a zwitterionic form and interact with guanosine via a ‘new wobble’ pattern.

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm(5)U) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) are located at the wobble position of human mitochondrial (hmt) tRNA(Leu(UUR)) and tRNA(Lys), respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys) 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 τm(5)U and τm(5)s(2)U into 17-mers related to the sequence of the anticodon arms hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys), 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 τm(5)s(2)U-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.

mt-tRNA components: synthesis of (2-thio)uridines modified with blocked glycine/taurine moieties at C-5,1.

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 (cmnm5(s2)U) and 5-taurinomethyl(-2-thio)uridine (τm5(s2)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.

An efficient approach for conversion of 5-substituted 2-thiouridines built in RNA oligomers into corresponding desulfured 4-pyrimidinone products

An efficient approach for the desulfuration of C5-substituted 2-thiouridines (R5S2U) bound in the RNA chain exclusively to 4-pyrimidinone nucleoside (R5H2U)-containing RNA products is proposed. This post-synthetic transformation avoids the preparation of a suitably protected H2U phosphoramidite, which otherwise would be necessary for solid-phase synthesis of the modified RNA. Optimization of the desulfuration, which included reaction stoichiometry, time and temperature, allowed to transform a set of ten R5S2U-RNAs into their R5H2U-RNA congeners in ca. 90% yield.

Escherichia coli tRNA 2‐selenouridine synthase (SelU) converts S2U‐RNA to Se2U‐RNA via S‐geranylated‐intermediate

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).