Brf1 is required for Ras-induced tRNA synthesis and growth in both wing imaginal discs and adult midgut progenitor cells (AMPs).

<p>(A). Ras^V12 expression was induced in <i>Drosophila</i> S2 cells for 24 hours in either control cells or Brf1 knockdown cells, Brf1 was knocked down by incubating cells with dsRNA against Brf1. Control cells were treated with dsRNA to GFP. Total RNA was isolated with Trizol and analyzed by northern blotting using DIG-labelled antisense probes to tRNA^iMet or tRNA^Arg. Ethidium bromide stained 5S rRNA band was used as a loading control. (B, C) <i>UAS-EGFR</i> and <i>UAS-Brf1 RNAi</i> were expressed, either alone or together, in the dorsal compartment of larval wing imaginal discs using an <i>ap-Gal4</i> driver. Control discs were from <i>ap-Gal4</i> crossed to <i>w</i>^<i>1118</i>. Wing discs were dissected at the wandering L3 larval stage and the area of the GFP-marked dorsal compartment quantified using NIH imaging software (n > 50 wings per genotype, data presented as mean +/- SEM). Representative images are shown in (B), quantification of tissue area is shown in (C). (D) <i>UAS-EGFR</i> and <i>UAS-Brf1 RNAi</i> were expressed, either alone or together, in the <i>Drosophila</i> larval AMPs using the <i>esg-Gal4</i>^<i>ts</i> system. Larvae were shifted to 29°C at 24 hrs of development to induce transgene expression and dissected as L3 larvae. AMPs are marked <i>by UAS-GFP</i> expression. DNA is stained with Hoechst dye (blue). (E) The number of cells in each AMP cluster was quantified for each of the genotypes in D (left), and an additional similar experiment in which the Ras pathway was activated by expression of a <i>UAS-Raf</i>^<i>gof</i> transgene (right). Data are presented as box plots (25%, median and 75% values) with error bars indicating the min and max values.