Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila

Abstract

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

Fig 1. Activated Ras/ERK signalling pathway stimulates protein synthesis.

(A) Left: Ras^V12 expression was induced in cultured Drosophila S2 cells for 24 hrs. Cells were then incubated in puromycin for 30 min. Protein extracts were separated by SDS-PAGE and analyzed by western blot with an antibody to puromycin to measure the levels of puromycin-labelled peptides. Cycloheximide treatment was for 15 mins prior to addition of puromycin. A phospho-ERK immunoblot is shown as an indication of Ras/ERK signalling pathway activation. An alpha-tubulin immunoblot is shown as a loading control. Right: Experiments were performed in at least three biological replicates and western blots were quantified using NIH Image J software. Data are represented as relative levels (mean +/- SEM) compared to control (B) A Representative polysome profiles from control S2 cells (blue) and S2 cells with induced Ras^V12 expression (red). Polysome peaks (arrowheads) in Ras^V12 expressing cells were higher compared to controls, suggesting translation was increased. (C) Left: Drosophila S2 cells were treated with in the presence or absence of 10 μM U0126 for 2 hours at 25°C. Cells were then incubated in puromycin for 30 min. Protein extracts were separated by SDS-PAGE and analyzed by western blot with an antibody to puromycin to measure the levels of puromycin-labelled peptides. A phospho-ERK immunoblot is shown as an indication of Ras/ERK signalling pathway activation. An alpha-tubulin immunoblot is shown as a loading control. Right: Experiments were performed in at least three biological replicates and western blots were quantified using NIH Image J software. Data are represented as relative levels (mean +/- SEM) compared to control. (D) Ras^V12 expression was induced in cultured Drosophila S2 cells for 48–96 hrs. Total protein content per 10³ cells was calculated using a Bradford assay. Data represent mean +/- SEM for at least three independent replicates per time point.

Fig 2. The Ras/ERK signalling pathway stimulates tRNA synthesis.

(A, B) Drosophila S2 cells were treated with 10 μM U0126 for 2 hours. Total RNA was isolated and levels of either pre-tRNAs (A), or total tRNAs (B) measured by qRT-PCR. N = 15 independent samples per condition. (C, D) Raf was knocked down in Drosophila S2 cells by incubating cells with dsRNAs against Raf. Control cells were treated with dsRNA to GFP. Total RNA was isolated and levels of either pre-tRNAs (C), or total tRNAs (D) measured by qRT-PCR. N = 4 independent samples per condition. (E, F) Ras^V12 expression was induced in Drosophila S2 cells for 24 hours. Total RNA was isolated and levels of either pre-tRNAs (E), or total tRNAs (F) measured by qRT-PCR. N = 9 independent samples per condition. (G) UAS-Raf^gof was expressed in imaginal tissues using the esg-GAL4^ts system. Control flies were esg-GAL4^ts flies crossed to w¹¹¹⁸. Transgenes were induced by shifting larvae to 29°C at 48hrs of larval development, and then discs were dissected from wandering L3 stage larvae. Total RNA was isolated and levels of pre-tRNAs measured by qRT-PCR. N = 4 independent samples per condition. Data are presented as mean +/- SEM.

Fig 3. Brf1 is required for Ras-induced tRNA synthesis and growth in both wing imaginal discs and adult midgut progenitor cells (AMPs).

(A). Ras^V12 expression was induced in Drosophila 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) UAS-EGFR and UAS-Brf1 RNAi were expressed, either alone or together, in the dorsal compartment of larval wing imaginal discs using an ap-Gal4 driver. Control discs were from ap-Gal4 crossed to w¹¹¹⁸. 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) UAS-EGFR and UAS-Brf1 RNAi were expressed, either alone or together, in the Drosophila larval AMPs using the esg-Gal4^ts system. Larvae were shifted to 29°C at 24 hrs of development to induce transgene expression and dissected as L3 larvae. AMPs are marked by UAS-GFP 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 UAS-Raf^gof transgene (right). Data are presented as box plots (25%, median and 75% values) with error bars indicating the min and max values.

Fig 4. Brf1 is required for intestinal stem cells (ISCs) homeostasis and for Ras-induced cell proliferation.

(A, B) UAS-Brf1 RNAi was expressed adult ISCs and EBs using the esg-GAL4^ts system. Control flies were esg-GAL4^ts flies crossed to w¹¹¹⁸. Flies were then fed with sucrose or sucrose plus DSS (A) or Bleomycin (B) for 2 days. Intestines were then dissected and stained for phospho-histone H3 positive cells. Data represent the mean number of phospho-histone H3 cells per intestine +/ SEM. N >15 intestines per condition. (C) A UAS-Ras^V12 transgene was expressed in adult intestines using the esg-GAL4^ts driver. Control samples (WT) expressed UAS-GFP alone. Total RNA was isolated and levels of pre-tRNAs measured by qRT-PCR. N = 4 independent samples per condition. Data are presented as mean +/- SEM. (D) UAS-Raf^gof and UAS-Brf1 RNAi were expressed, either alone or together, in the adult ISCs and EBs using the esg-Gal4^ts system. esg positive cells are marked with GFP and DNA is stained with Hoechst dye. Knockdown of Brf 1(UAS-Brf RNAi) suppresses the increased proliferation seen with UAS-Raf^gof expression.

Fig 5. dMyc is required but not sufficient for Ras-induced tRNA synthesis.

(A) Ras^V12 expression was induced in Drosophila S2 cells for 24 hours in either control cells or dMyc knockdown cells. dMyc was knocked down by incubating cells with dsRNA against dMyc. 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 and D) dMyc expression was induced in S2 cells for 24hrs, and then cells were treated with 10 μM U0126 or DMSO for 2 hours. Total RNA was isolated with Trizol and analyzed by qRT-PCR to measure levels of (B) pre-tRNAs, (C) dMyc mRNA, or (D) mRNA levels of three dMyc target genes—NOP60B, PPAN and NOP5. N = 4 independent samples per condition. Data are presented as mean +/-SEM.

Fig 6. Ras induces tRNA synthesis by via inhibition of the Pol III repressor, dMaf1.

(A, B) dMaf1 was knocked down in Drosophila S2 cells by incubating cells with dsRNAs against dMaf1. Control cells were treated with dsRNA to GFP. Cells were then treated with DMSO (control) or 10 μM U0126 for 2 hrs. Total RNA was isolated and levels of pre-tRNATyr (A) and pre-tRNALeu measured by qRT-PCR. N = 4 independent samples per condition. Data are presented as mean +/-SEM. (C) dMaf1 subcellular localization was assessed by immunostaining with an anti-dMaf1 antibody in both control and 10 μM U0126 treated S2 cells. Green: dMaf1 staining; blue: Hoechst-stained nuclei. (D) The differential localization of dMaf1 in S2 cells from the experiment shown in (C) was quantified by measuring the ratio of nucleus: cytoplasmic intensity quantified using NIH Image J. Data are plotted in the graph as relative nuclear: cytoplasmic staining intensity for both experimental conditions. Data are presented as box plots (25%, median and 75% values) with error bars indicating the min and max values. N>90 cells per condition fom three independent experiments. (E) A UAS-Ras^N17 transgene was expressed in GFP-marked adult midgut progenitor cells using the esg-GAL4^ts driver. Control samples (WT) expressed UAS-GFP alone. dMaf1 subcellular localization was then assessed in third instar larvae by immunostaining with an anti-dMaf1 antibody. (F) The differential localization of dMaf1 in the AMPs from the experiment shown in (E) was quantified by measuring the ratio of nucleus: cytoplasmic intensity quantified using NIH Image J. Data are plotted in the graph as relative nuclear: cytoplasmic staining intensity for both experimental conditions. Data are presented as box plots (25%, median and 75% values) with error bars indicating the min and max values. N>30 cells per condition fom three independent experiments.

Fig 7. Ras signalling regulates dMaf1 phosphorylation.

(A) Drosophila S2 cells were treated with 10 μM U0126 for 2 hours. Cells were then lysed and processed for SDS-PAGE and western blotting using the phos-tag reagent, as described in the Methods. The blots were then probed with an anti-dMaf1 antibody (top panel), an anti-total ERK antibody (middle panel) or an anti-phospho ERK antibody (lower panel) (B) Ras^V12 expression was induced in Drosophila S2 cells for 24 hours. Cells were then lysed and processed for SDS-PAGE and western blotting using the phos-tag reagent, as described in the Methods. The blots were then probed with an anti-dMaf1 antibody (top panel), an anti-total ERK antibody (middle panel) or an anti-phospho ERK antibody (lower panel). (C) A model for how Ras signalling may regulate Pol III and tRNA synthesis.