Figure 4. The Methylated tRNA Speices are Preferrentially Used to Promote Translation.

(A) Hypermethylated tRNA^Val(mAC), tRNA^His(GUG), and tRNA^Gly(GCC) preferentially associate with the polysomes. Quantification of the m¹A/G ratio in tRNA^Val(mAC), tRNA^His(GUG), and tRNA^Gly(GCC) extracted from the translationally inactive portions (< 80S) and the translationally active portions (> 80S) in ALKBH1 stable overexpression HeLa cells and the control cells. Error bars represent mean ± s.d., n = 8 (four biological replicates × two technical replicates).

(B) The elongation factor-1 protein (eEF1α) was pulled down from HeLa cells using a specific anti-eEF1α antibody and then incubated with an excess amount of total tRNA purified from same cells. LC-MS/MS measurements showed enrichment of m¹A in the eEF1α-bound portion and depletion of m¹A in the flow through portion. Error bars represent mean ± s.d., n = 8 (four biological replicates × two technical replicates).

(C) Quantification of protein synthesis in MEF cells by flow cytometry. HPG incorporation into MEF control cells (grey), Alkbh1^−/− MEF cells (red), HeLa control cells (yellow), and ALKBH1 stable overexpression HeLa cells (blue) were recorded 1 h after administration. Six biological replicates were performed; for ALKBH1 stable overexpression compared to the control, p = 0.012. For Alkbh1^−/− MEF vs. control, p = 0.003. P values were determined using two-tailed Student’s t-test for paired samples. *p < 0.05, **p < 0.01. Error bars represent mean ± s.d., n = 9 (three biological replicates × three technical replicates).

See also Figure S4.