Figure 2.
The CCA-adding enzyme repairs trimmed tRNAs. (A) The levels of an in situ HA-tagged CCA-adding enzyme (CAE-HA) were investigated by western blot on cells growing exponentially or upon nutritional stress. EF1A serves as loading control. (B) The tagged CAE was purified from parasites growing exponentially (expo) or subjected to nutritional stress (stress) and its activity was tested using tRNAs purified from stressed cells and α32P-CTP after 5, 15 and 30 min of incubation (upper panel). A mock purification was done using cells not expressing the tagged CAE. A representative gel is shown. The levels of the CAE used were analyzed by western blot (lower panel). (C) Signals from experiments in B were quantified and normalized to the intensity of the product generated by the CAE purified from exponentially growing cells and incubated for 5 min. Depicted is the mean of 3 independent experiments ± SD. (D, E) Cells allowing inducible overexpression (D) or depletion (E) of the CAE were stressed and then allowed to recover in normal media. Uninduced cells (−) exposed to the same conditions serve as control. RNA was extracted at the indicated time points and shortening of tRNAVal was analyzed by northern blot. Full length tRNAs and 3′ trimmed tRNAs are indicated with filled and open arrowheads, respectively. The percentage of full length tRNAVal present is indicated (% FL).