Enzymatic conversion of adenosine to inosine and to N1-methylinosine in transfer RNAs: A review

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

“…In eukaryotes, the tRNA Ile that reads exclusively the AUA codon has a UAU anticodon which, at least in S. cerevisiae, is modified to cAc (tRNA Ile CAC ; c, pseudouridine) (Szweykowska-Kulinska et al 1994). Another tRNA Ile with the anticodon IAU (tRNA Ile IAC ; I, inosine) can also read the AUA codon in addition to the major isoleucine codons AUU and AUC (Crick 1966;Grosjean et al 1996;Senger et al 1997 Ile from H. volcanii and H. salinarum (Fig. 6B) and lack of reaction of H. volcanii, H. salinarum, and M. jannaschii tRNAs with an NHS-ester specific for primary amino groups (data not shown) suggest that these tRNAs may also contain the same modified C34.…”

tRNA base methylation identification and quantification via high-throughput sequencing

“…5). AlaAGC is known to contain two inosine modifications at 34 (first anticodon nucleotide) and 37, and three W-C face methylations at m 1 A58, m 1 I37, and m 2 2 G26 (Grosjean et al 1996;Machnicka et al 2013). I34 is readily apparent in the mutation graph as essentially 100% of this residue has been converted from A to I, which reads as G, and it does not respond to demethylase treatment (Fig.…”

Inosine and N 1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNAAla are major epitopes for anti-PL-12 myositis autoantibodies

“…Several autoantibodies that recognize various deproteinized RNAs have been described (reviewed in Hoet & Van Venrooij, 1992;Keene, 1996;Mimori, 1996)+ Among them, only those against U1-U5 snRNA, as detected in few patients with systemic sclerosis, were shown to be directed to a specific modified nucleoside (2,2,7-trimethylguanosine cap structure) (Okano & -methylinosinic acid (pm 1 I) are higher in F than in E because the ratio between modified and nonmodified nucleotides in the variant RNA 7 is higher than for variant RNA 5+ The solvent system 1 as described in Auxilien et al+ (1996) was used in all cases+ Medsger, 1992)+ Except for the autoantibodies against tRNA Ala (anticodon IGC) described in the present study, the other anti-RNA autoantibodies characterized so far are not directed to modified nucleotides, as they also immunoprecipitate in vitro transcripts, lacking modified nucleosides, of the natural or synthetic genes corresponding to these antigenic RNAs+ Examples include autoantibodies against a characteristic 40-nt stem-loop fragment in U1snRNA (Deutscher & Keene, 1988;Tsai et al+, 1992;Teunissen et al+, 1998), a 59-nt fragment of 28S rRNA (Chu et al+, 1991), a 84-nt fragment of Y5 Ro RNA (Boulanger et al+, 1995), or various mutants of tRNA retaining their L-shaped three-dimensional architecture (Matsumura et al+, 1996;Brouwer et al+, 1998;Ohosone et al+, 1998)+ In these cases a few selected canonical nucleotides within a well-defined RNA conformation (a characteristic stem-loop or a three-dimensional architecture) constitute the major epitopes recognized by the corresponding IgG+ It is worth mentioning that antibodies predominantly directed towards the characteristic hypermodified nucleoside wybutosine, which is located at position 37 of the anticodon loop of yeast tRNA Phe , were obtained by immunizing a goat with a glutaraldehyde-mediated conjugate of yeast tRNA Phe and bovine gamma globulin (Fuchs et al+, 1974)+ Also, antibodies against a variety of modified nucleosides, including inosine (Inouye et al+, 1971) and 1-methylinosine (D'Ambrosio et al+, 1991; Renaud et al+, 1991) have been prepared by immunizing rabbits, mice or goats with a conjugate of the hapten and a protein carrier (reviewed in Vold, 1990)+ In this article, we present evidence that the important features recognized by human anti-tRNA Ala (anti-PL-12) autoantibodies include the chemical identity and the spatial disposition of two modified ribonucleotides within the anticodon loop of tRNA Ala (anticodon IGC): namely the bases inosine at position 34 and N 1 -methylinosine at position 37 (see Fig+ 1A), their spacing, and probably their three-dimensional configuration+ Figure 5 shows the spatial orientation of the 6-keto groups (in black) of inosine-34 and of the N 1 -methyl group (in grey) of N 1 -methylinosi...…”

“…The length of each oligonucleotide was checked by 15% (w:v) polyacrylamide gel electrophoresis in the presence of 8 M urea+ Their migrations were compared to oligodeoxynucleotide size markers (8-32 mers: Pharmacia, Uppsala, Sweden)+ Prior to electrophoresis, an aliquot of each type of oligonucleotide was radiolabeled as described above+ The identity of the 59 terminal nucleotide of each newly chemically synthesized oligonucleotide was also verified+ To this end, the [ 32 P]-oligonucleotides recovered from the gel were completely hydrolyzed with nuclease P 1 + The resulting hydrolyzates were analyzed by two-dimensional thin-layer chromatography using the solvent system 1 as described elsewhere (Auxilien et al+, 1996)+ After migration, the plates were autoradiographed and the 59-[…”

“…In previous work, we demonstrated that modified nucleosides in the anticodon of yeast and Bombyx mori tRNA Ala are indeed major epitopes for anti-tRNA Ala autoantibodies (Y+ Corda & H+ Grosjean, 1995, and in prep+)+ This was accomplished using transcripts from synthetic tRNA Ala genes with nucleotide sequences identical (at least in the anticodon stem and loop regions) to that of human tRNA Ala (anticodon AGC)+ These transcripts, produced in vitro by T7 RNA polymerase, lacked modified nucleosides and failed to precipitate with human antitRNA Ala autoantibodies+ The tRNA transcripts became strongly reactive to anti-tRNA Ala autoantibodies when both adenosine-34 and adenosine-37 were enzymatically converted into inosine-34 and N 1 -methylinosine-37, respectively, by incubation with an enzyme extract from yeast or rat liver+ However, this enzymatic approach has limitations imposed by the specificities of the modifying enzymes, which are very much dependent on tRNA architecture (Auxilien et al+, 1996;Grosjean et al+, 1996;Gerber et al+, 1998)+ Therefore, it was not possible to test systematically whether antibody recognition is sensitive to a reduction in the size of the RNA molecule, to variations in the relative positions of inosine and N 1 -methylinosine in the RNA sequence, or to the presence of deoxy-analogs of the anticodon stem-loop nucleosides+…”