Figure 2.
DmSRS2 knockdown strength. (A) DmSRS2 mRNA levels were quantified by real-time PCR in control larvae (w1118) and RNAiDmSRS2 larvae from strains 1-dcr2 and 23003 crossed with actin5C-GAL4 driver at 29 and 25°C, respectively. DmSRS2 mRNA levels were normalized using Rp49 mRNA as reference. Graph gives average with SEM from three independent experiments, and statistical significance is calculated by Student’s t-test (*P < 0.05; ***P < 0.001). The mRNA level in control larvae is established as 1 and the other values are relative to this. (B) Aminoacylated and deacylated mitochondrial tRNAs (mt-tRNAs) were detected by northern blot from larvae with inactive (−) and active (+) RNAi for DmSRS2, coming from strain 1-dcr2 and 23003 crossed with actin5C-GAL4 driver at 29 and 25°C, respectively. Thirty micrograms of total RNA were loaded into high-resolution acid gels, and probes were designed to specifically detect the two mitochondrial tRNASer isoacceptors (GCU and UGA) and the mitochondrial tRNALys (CUU) as control. (C) The graph shows, for each lane from panel B, the relative abundance of aminoacylated mt-tRNALys (CUU) (black), mt-tRNASer (GCU) (gray) and mt-tRNASer (UGA) (white), setting the levels of aminoacylated mt-tRNAs in larvae with inactive (−) RNAi as 100%. The mean from two independent experiments with SEM are represented and subjected to Student’s t-test (*P < 0.05; **P < 0.01).