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. 2012 Oct 5;40(22):11583–11593. doi: 10.1093/nar/gks910

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Loss of tRNA:m¹G9 and tRNA:m¹A9 methyltransferase activity in HeLa cell mitochondrial extracts after RNAi-mediated knock-down of TRMT10C or SDR5C1 mRNA. Cells were transfected with a control siRNA or with two different gene specific siRNAs, respectively. Forty-eight hours after transfection, total RNA was extracted from a sample, and relative TRMT10C and SDR5C1 mRNA levels were determined by quantitative real-time RT-PCR and were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (control siRNA, 116%; TRMT10C siRNA#1, 33%; TRMT10C siRNA#2, 18%; control siRNA, 119%; SDR5C1 siRNA#1, 14%; SDR5C1 siRNA#2, 4%). Based on previously established knock-down kinetics, mitochondrial extracts were prepared 3 days after transfection in the TRMT10C RNAi experiment and 6 days after transfection in the SDR5C1 RNAi experiment (17). Extracts were adjusted for their protein concentration. (A) tRNA:m¹G9 methyltransferase activity in mitochondrial extracts of siRNA-treated HeLa cells. Position 9-labelled (mt)tRNA^Tyr was used as substrate, and the tRNA hydrolysate was resolved by TLC. (B) tRNA:m¹A9 methyltransferase activity in mitochondrial extracts of siRNA-treated HeLa cells. Position 9-labelled (mt)tRNA^Lys was used as substrate. The middle part of the TLC was cropped as indicated and only the informative part is shown.