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. 2012 Jan 20;287(12):8892–8903. doi: 10.1074/jbc.M111.300186

FIGURE 3.

FIGURE 3.

An in vivo assay for tRNA import in trypanosomes. A, shown is doxycycline-regulated expression of Var-tRNAMet-i in T. brucei. Total cell RNA from 2 × 107 trypanosomes were grown with (+) or without (−) the addition of doxycycline (1 μg/ml) for 24 h. RNA was fractionated by non-denaturing polyacrylamide gel electrophoresis (6%) (top panel), and abundant rRNA and tRNA were visualized by ethidium bromide (EtBr) staining. The Var-tRNAMet-i was detected by Northern blot hybridization and autoradiography (bottom panel). B, shown is mitochondrial import of Var-tRNAMet-i after doxycycline induction. Total cellular and mitochondrial RNA was isolated 24 h post-induction with doxycycline in three independent experiments. Samples were fractionated on 6% polyacrylamide gel, stained with EtBr to visualize the mitochondrial 9 S and 12 S rRNAs, small mature cytosolic rRNAs, and tRNAs (upper panel), and hybridized with a probe for Var-tRNAMet-i (lower panel). C, total cellular (TC) and mitochondrial (M) RNA from control (PC 29−13) was isolated and fractionated by non-denaturing polyacrylamide gel electrophoresis (8%) (top panel), and abundant rRNAs and tRNAs were visualized by ethidium bromide staining. The cytosolic-specific WT tRNAMet-i was detected by Northern blot hybridization and autoradiography. D, shown is quantitative analysis of Var-tRNAMet-i import at 24 h post induction with doxycycline (n = 8). The percentage of tRNA import into the mitochondrion was calculated based on the hybridization of the Var-tRNAMet-i probe to total cellular RNA and RNA extracted from purified mitochondria. E, shown is analysis of tRNA import after RNAi knockdown of eEF1a or eIF2. Samples were fractionated on 6% polyacrylamide gels, stained with EtBr (upper panel) total cellular (TC), and mitochondrial (M) RNA were analyzed by Northern blot (lower panel) for amount of Var-tRNAMet-i imported into mitochondria. F, quantitative analysis of tRNA import for eEF1a (n = 6) and eIF2 (n = 3) was expressed relative to the average level of Var-tRNAMet-i import in wild type cells (10.3%, n = 8).