FIGURE 2.

Tcs3 is required for t⁶A synthesis in Drosophila. A, yeasts were plated in solid media in serial dilutions of 1:10 factor from left to right. Growth differences of wild-type, TCS3 mutant cells (Δtcs3), and mutant yeasts expressing the Drosophila Tcs3 homolog (Δtcs3+Tcs3). Growth parameter of each strain was also analyzed in liquid media. Each strain has a color code (n = 10, p < 0.005). B, in Drosophila, Tcs3 was knocked-down using a specific inverted repeat construct expressed in the posterior wing compartment with the Gal4/UAS system (Tcs3-IR) and the growth phenotype evoked was rescued by yeast Tcs3p co-expression (en> Tcs3-IR+Tcs3). The area of sector D (colored in green) was measured and plotted following the same color code used in wing image labels (n = 50, mean ± S.D., t test p < 0.005). C, PHAt⁶A. In brief, probes designed against different regions of the initiator tRNA evidenced the presence of t⁶A modification. The strength of ASL probe (anti-codon stem loop) hybridization depends on the presence of t⁶A, whereas the TΨL probe hybridizes an unmodified base stretch and serves as an internal loading control. When t⁶A is absent, the tRNA-probe interaction is maximal, whereas its existence weakens the interaction. D, tRNAs from wild-type (wt) and mutant tcs3 animals (tcs3^e and tcs3^f) were probed with ASL and TΨL probes. Dot blots were merged to make a better composition. Also a plot depicting the change in the ASL/TΨL signal ratio is shown. ASL signals obtained from tcs3 mutants are significantly stronger than control counterparts (n = 6 samples).