Skip to main content
. 2012 Dec;18(12):2269–2276. doi: 10.1261/rna.035600.112

FIGURE 3.

FIGURE 3.

Detection of hypomodified m1A58 in mitochondrial tRNAs from siRNA-treated cells. After knockdown of luciferase (control), Trmt61B, or Trmt6 mRNAs, primer extension was used to detect methylated or nonmethylated A58 in mitochondrial tRNALeu(UUR) (A), mitochondrial tRNASer(UCN) (B), mitochondrial tRNALys (C), and cytoplasmic tRNAHis (D). Two different siRNAs (si1 and si2) targeting Trmt61B mRNA were used. In A, “+” indicates that the cells were transfected once with siRNA and harvested 4 d after transfection. “++” indicates that the cells were transfected with siRNA a total of three times (every 2 d) and harvested 6 d after the first transfection. In B–D, cells were transfected once with siRNA and harvested 4 d after transfection. The knockdown efficiencies of Trmt61B and Trmt6 mRNAs were quantified by qRT-PCR and normalized to GAPDH mRNA. The steady-state level of Trmt61B mRNA was decreased to 8.0% (si1, +), 8.3% (si1, ++), 18% (si2, +), and 12% (si2, ++), and Trmt6 mRNA was decreased to 17% (si-Trm6, +) and 17% (si-Trm6, ++) compared with si-Luc cells. The primers are shown as solid lines next to the tRNAs, and nascent cDNAs synthesized from the primers are depicted as gray lines.