Fig. 5.

Analysis of modifications at the first nucleotide of the anticodons in human tRNA^Val(UAC), tRNA^Arg(UCU), and tRNA^Gly(UCC) from both FD carrier and patient cells. (A, Left) Scheme for tRNA modification analysis of the anticodon loop using reciprocal circulating chromatography (RCC) and liquid chromatography/mass spectrometry (LC/MS) measurements. AEC, anion exchange chromatography. (A, Right) ELPs are likely involved in the step for ncm⁵U synthesis. Modified residues are highlighted by the colors consistent with those colors used for chromatograms and histograms shown in B–D. (B–D, Top) Secondary structures and sequences at the anticodon stem–loop region of indicated tRNAs are shown. The wobble uridine residue of each tRNA is indicated with possible modifications. The reported modifications m³C and t⁶A near the anticodon are also indicated. The sequences indicated in bold letters are the RNase T₁ fragments harboring the anticodons. (B–D, Middle) LC/MS analyses of the RNase T₁ fragments of each tRNA from FD carrier (Carrier) and patient (#42 and #50) cells are shown. Each arrow with the number corresponding in Table S2 indicates the position of the peak of the mass chromatogram of each RNase T₁ fragment. All sequences of RNase T₁ fragments analyzed by LC/MS are listed in Table S2. (B–D, Bottom) Histograms show the percentage of modifications in total uridine residues at the wobble position for each tRNA.