Effect of multiple Dhh1 mutations on cell growth and on viral RNA translation. (A) Motifs and functional domains of the DEAD-box RNA helicase Dhh1. Numbers 1A to 6B indicate the location of the point mutations. (B) Growth at 30°C and 36°C of WT and dhh1Δ expressing various Dhh1 mutant alleles. (C) Effect of Dhh1 mutants on BMV RNA2 translation. The dhh1Δ strain was transformed with a plasmid expressing RNA2 together with an empty plasmid or a plasmid expressing DHH1 or the different dhh1 mutant alleles. RNA2 and protein 2a were analyzed by Northern and Western blot, respectively. As a control for equal loading and sample quality, 18S rRNA and phosphoglycerate kinase protein 1 (Pgk1) were also analyzed. The average 2a expression value from at least three independent experiments is shown. The average value obtained for cells expressing DHH1 was set to 100. The SEM values are indicated below. (D) Dhh1 binds BMV RNA2. Western blot analysis of immunoprecipitation assays carried out in dhh1Δ cells expressing RNA2 and Dhh1 or Dhh1-Flag. Input corresponds to 100 µg of total protein present in lysates prior to precipitation and IP corresponds to the corresponding eluates from the anti-Flag matrix following precipitation. Diagram shows the relative amount (±SEM) of precipitated RNA2 detected by qPCR after immunoprecipitation; the input amount of RNA2 was set to 100. (E) The half-life of RNA2 is not significantly altered in dhh1Δ cells. RNA2 was expressed from a GAL1 promoter in WT and dhh1Δ cells. Transcription was stopped by adding glucose at time point t = 0 min. Samples were taken at different time points and RNA2 accumulation analyzed by Northern blot. Numbers below indicate the average half-life of RNA2 (±SEM) based on three independent experiments. T-test analysis shows that the half-life is not significantly altered in dhh1Δ cells.
