Figure 3.

TRMT2B catalyses m⁵U54 in mt-tRNA^Pro.

(A) Schematic of mt-tRNA^Pro T-loop showing annealed primer to be extended (red line) and the position of m⁵U54 (blue text). HeLa cell derived RNA, either following a 6-day siRNA mediated depletion of TRMT2B or untreated, was subsequently either untreated (-) or treated with hydrazine for either 5 or 10 minutes, followed by aniline, to specifically cleave at unmodified uridine residues. This RNA was subjected to RT-PEx using a [32P]-end labelled primer complementary to the region upstream of m⁵U54 (red line). The nucleotide sequence of the tRNA, corresponding to stalling events at each position, is shown to the side of the panel. Quantification values represent the ratio between stalling at U54 and stalling at the next uridine residue (U49), after the values in the untreated lanes had been subtracted from both to account for background. (B) RT-PEx reactions as performed above with RNA derived from a HAP1 parental cell line with wild-type (WT) or a HAP1 TRMT2B knockout cell line (KO). Error bars = SEM, n = 3.