Maf1-mediated repression of RNA polymerase III transcription inhibits tRNA degradation via RTD pathway

Partial inactivation of Pol III subunit results in suppression of trm4Δtrm8Δ ts phenotype and tRNA^Val(AAC) stabilization. (A) Cold-sensitive rpc128-1007 mutation in Pol III subunit Rpc128 was introduced to trm4Δtrm8Δ strain by genetic cross, resulting in triple trm4Δtrm8Δrpc128-1007 mutant. Serial 10-fold dilutions of BY4741 (wt), rpc128-1007, trm4Δtrm8Δ, and trm4Δtrm8Δrpc128-1007 cells were spotted on YPD plates and incubated at indicated temperatures. (B) Cells were grown in YPD to OD₆₀₀ = 1.0 at 30°C, transferred to 37°C, and harvested as indicated. RNA was isolated and examined by Northern hybridization with probe specific for tRNA^Val(AAC). Plots represent levels of tRNA^Val(AAC) normalized to loading control (5.8S rRNA). The magnitude of change was calculated relative to expression in trm4Δtrm8Δ strain at 0 time point. SD was calculated on the basis of three independent experiments. P-values—P < 0.01 for the probe indicated with a single asterisk (*) and P < 0.005 for the probe indicated with two asterisks (**)—were calculated relative to control strain at each time point. (C) Gene encoding Rpc17 subunit of Pol III was cloned in trm4Δtrm8Δ strain under the regulated tetO₇ promoter to allow down-regulation of RPC17 expression by doxycycline. Serial 10-fold dilutions of trm4Δtrm8Δ tetO₇∷RPC17 and control cells were spotted to YPD plate containing 5 μg/mL of doxycycline and incubated for 2 d at indicated temperatures. Phenotypic suppression of the ts phenotype trm4Δtrm8Δ was most efficient at 36°C. (D) trm4Δtrm8Δ tetO₇∷RPC17 cells were grown in YPD with or without 2 μg/mL of doxycycline to OD₆₀₀ = 1.0 at 23°C, transferred to 37°C, and harvested as indicated. RNA was isolated and examined by Northern hybridization with probe specific for tRNA^Val(AAC). Plots represent levels of RNA^Val(AAC) normalized to loading control (5.8S rRNA). The magnitude of change was calculated relative to expression in culture without doxycycline at zero (0) time point. SD was calculated on the basis of three independent experiments. P-values—P < 0.005 for the probes indicated with two asterisks (**)—were calculated relative to control strain at each time point.