Fig. 6: Validation of 5-EC-iCLIP tRNA hits.
From: Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases
a Strategy for LC-QQQ-MS quantification of modified nucleosides in individual tRNAs isolated from HEK 293T wild-type (WT) and ALKBH1 KO cells. Individual tRNAs are enriched by antisense pulldown, digested to mononucleosides and analyzed by LC-QQQ-MS. b–g Quantification of oxidized m5C products in b mt-tRNA-Met, p-values: WT vs KO for m5C, 0.026079; for f5C, 0.000132; for hm5C, 0.000244, c tRNA-Leu-CAA, p-values: WT vs KO for m5C, 0.156295; for f5C, 0.000170; for hm5C, 0.000002, d tRNA-Glu-CTC, p-values: WT vs KO for m5C, 0.975934; for f5C, 0.002584, e tRNA-Gly-CCC, p-values: WT vs KO for m5C, 0.161660; for f5C, 0.001039, f tRNA-Gln-CTG, p-values: WT vs KO for m5C, 0.365015; for f5C, 0.171454, and g tRNA-Val-CAC, p-values: WT vs KO for m5C, 0.152377; for f5C, 0.124395; for hm5C, 0.366551. h Schematic workflow for pyridine borane f5C sequencing. Pyridine borane converts f5C residues to dihydrouridine, which is read as uridine, thus generating a C-to-T signature that can be identified in sequencing (i) Validation of presence and ALKBH1-dependence of f5C at the wobble base of mt-tRNA-Met by pyridine borane sequencing. j Validation of presence and ALKBH1-dependence of f5C at the wobble base of tRNA-Leu-CAA. k Presence and ALKBH1-dependence of f5C at various positions of tRNA-Val-CAC and tRNA Glu-CTC. For (b)–(g), three independent biological replicates were analyzed. Data represent mean values ± s.d. An unpaired t-test (two-tailed) was used to measure the statistical significance *p  <  0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For (h)–(j), two independent biological replicates were performed and analyzed. Source data are provided as a Source Data file.
