Analysis of t6A-modified tRNAs in yeast KEOPS and Drosophila kae1 mutants. (A) Analysis of yeast t6A pathway mutants is shown as a control. Primer extension assays yield a ladder of bands indicating the presence of stalled products. These include a band at position A37 of tRNA-Ile[AAU] of wild-type yeast that is strongly depleted in yeast t6A pathway mutants, thus indicating a t6A-modified nucleoside. tRNA-Val[UAC] is shown as a control for a non-t6A-bearing substrate. The ladder positions are inaccurate due to incomplete digestion; therefore, sizes are labeled according to the input primer. (B) Analysis of Drosophila kae1 mutants. Modifications of the A37 positions of tRNA-Met[CAU] and tRNA-Ile[AAU] are decreased although not eliminated in two strong hemizygous conditions of kae1. The reduction observed in kae1[1A13]/Df was rescued by a P[acman]-kae1 genomic transgene (“kae1+”). Note that mutant larvae were collected at an advanced age in their extended larval period to separate them from potential maternal contributions; the normal larval period ends at ∼5 d. (C) LC-MS/MS analysis of total tRNAs from Drosophila kae1 hemizygous mutant larvae. We observe a minor decrease in total t6A-modified tRNAs in the hypomorphic allele [7A9] and stronger decreases in the strong/null alleles [f01978] and [1A13]. The levels of t6A-modified tRNAs did not decrease further in exceptionally long-lived kae1 mutant larvae, and the loss of t6A was rescued by a kae1 genomic transgene.
