tRNA's wobble decoding of the genome: 40 years of modification.

Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.

Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Post-transcriptional ribonucleotide modification is a phenomenon best studied in tRNA, where it occurs most frequently and in great chemical diversity. This paper reviews the intrinsic network of modifications in the structural core of the tRNA, which governs structural flexibility and rigidity to fine-tune the molecule to peak performance and to regulate its steady-state level. Structural effects of RNA modifications range from nanometer-scale rearrangements to subtle restrictions of conformational space on the angstrom scale. Structural stabilization resulting from nucleotide modification results in increased thermal stability and translates into protection against unspecific degradation by bases and nucleases. Several mechanisms of specific degradation of hypomodified tRNA, which were only recently discovered, provide a link between structural and metabolic stability.

tRNA stabilization by modified nucleotides.

Alternative foldings are an inherent property of RNA and a ubiquitous problem in scientific investigations. To a living organism, alternative foldings can be a blessing or a problem, and so nature has found both, ways to harness this property and ways to avoid the drawbacks. A simple and effective method employed by nature to avoid unwanted folding is the modulation of conformation space through post-transcriptional base modification. Modified nucleotides occur in almost all classes of natural RNAs in great chemical diversity. There are about 100 different base modifications known, which may perform a plethora of functions. The presumably most ancient and simple nucleotide modifications, such as methylations and uridine isomerization, are able to perform structural tasks on the most basic level, namely by blocking or reinforcing single base-pairs or even single hydrogen bonds in RNA. In this paper, functional, genomic and structural evidence on cases of folding space alteration by post-transcriptional modifications in native RNA are reviewed.

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Post-transcriptional nucleotide modification and alternative folding of RNA.

Transfer RNA (tRNA) from all organisms on this planet contains modified nucleosides, which are derivatives of the four major nucleosides. tRNA from Escherichia coli/Salmonella enterica contains 31 different modified nucleosides, which are all, except for one (Queuosine[Q]), synthesized on an oligonucleotide precursor, which through specific enzymes later matures into tRNA. The corresponding structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The syntheses of some of them (e.g.,several methylated derivatives) are catalyzed by one enzyme, which is position and base specific, but synthesis of some have a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N6-threonyladenosine [t6A],and Q). Several of the modified nucleosides are essential for viability (e.g.,lysidin, t6A, 1-methylguanosine), whereas deficiency in others induces severe growth defects. However, some have no or only a small effect on growth at laboratory conditions. Modified nucleosides that are present in the anticodon loop or stem have a fundamental influence on the efficiency of charging the tRNA, reading cognate codons, and preventing missense and frameshift errors. Those, which are present in the body of the tRNA, have a primarily stabilizing effect on the tRNA. Thus, the ubiquitouspresence of these modified nucleosides plays a pivotal role in the function of the tRNA by their influence on the stability and activity of the tRNA.

Transfer RNA Modification

In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].

Synthesis and Studies on the Effect of 2-Thiouridine and 4-Thiouridine on Sugar Conformation and RNA Duplex Stability

Previous nuclear magnetic resonance (NMR) studies of unmodified and ψ39-modified tRNALys anticodon stem loops (ASLs) show that significant structural rearrangements must occur to attain a canonical anticodon loop conformation. The Escherichia coli tRNALys modifications mnm5s2U34 and t6A37 have indeed been shown to remodel the anticodon loop, although significant dynamic flexibility remains within the weakly stacked U35 and U36 anticodon residues. The present study examines the individual effects of mnm5s2U34, s2U34, t6A37, and Mg2+ on tRNALys ASLs to decipher how the E. coli modifications accomplish the noncanonical to canonical structural transition. We also investigated the effects of the corresponding human tRNALys,3 versions of the E. coli modifications, using NMR to analyze tRNA ASLs containing the nucleosides mcm5U34, mcm5s2U34, and ms2t6A37. The human wobble modification has a less dramatic loop remodeling effect, presumably because of the absence of a positive charge on the mcm5 side chain. Nonspe...

Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A.

Synthesis of Oligoribonucleotides Containing 2-Thiouridine: Incorporation of 2-Thiouridine Phosphoramidite without Base Protection

A pentamer RNA sequence, Gs2UUUC, and a s2U containing 14-mer RNA tetraloop hairpin were synthesized and characterized by NMR and by UV melting studies. These oligonucleotides were used as models to understand the effect of 2-thiouridine substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through sU-34 modification. The magnitude of the effect of sU in our model system is comparable to the 20° C stabilization reported for 2-thiolation in a codonanticodon model system composed of two tRNAs with complementary anticodon sequences.

Raju K. Kumar

Papers

Synthesis and Studies on the Effect of 2-Thiouridine and 4-Thiouridine on Sugar Conformation and RNA Duplex Stability

Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A.

Synthesis of Oligoribonucleotides Containing 2-Thiouridine: Incorporation of 2-Thiouridine Phosphoramidite without Base Protection

Structural studies of 2-thiouridine in rna

Effect of Sm3+ and Yb3+ doping on the structural and magnetic properties of La2−xRExNiMnO6 (x = 0.1–0.7) double perovskites