Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila

Abstract

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth.

Figure 1

Loss of Brf function leads to severe growth defects in Drosophila larvae. (A) Brf protein levels were reduced in brf mutant (brf^EY02964) larvae compared with controls (yw) 48 h after egg laying (AEL), as determined by immunoblot. (B) Levels of Pol III-dependent transcripts were significantly decreased in brf mutant larvae compared with control larvae 48 h AEL, as measured by qRT–PCR (P<0.05, Student's t-test). Error bars indicate s.e.m. (C) brf mutant larvae are growth arrested. Images of control and brf mutant larvae throughout larval development (48–120 h AEL) are shown. (D) brf clones (non-GFP, arrowheads) were induced by flp/FRT-mediated recombination during embryogenesis and visualized in the fat body 120 h AEL. Blue, DAPI staining; red, actin; green, GFP. Scale bar, 100 μm. (E) brf mutant clones in wing discs were induced 72 h AEL and clone areas measured 120 h AEL (n=100 twin spots). (F) brf mutant clones were induced and scored in wing imaginal discs at the times indicated post clone induction. The viability of mutant clones was assessed by counting the percentage of wild-type clones that were still paired with a brf mutant twin spot. Genotypes used in (D–F): hsflp¹²²;+; FRT82B, brf^EY02964/FRT82B, ubi-GFP.

Figure 2

Fat body-specific reduction in Brf activity has cell non-autonomous effects on organismal growth and development. (A) Fat body-specific reduction in Brf levels (r4>brf RNAi, green trace) delayed pupariation when compared with controls (r4>+, black trace). The data are represented as a percentage of larvae that develop to pupal stage. Error bars indicate s.e.m. (B) Fat body loss of Brf function results in smaller wing imaginal discs, compared with controls (n>20 per genotype). Disc size was quantified in wandering third instar larvae using the histogram tool (Adobe Photoshop). Error bars indicate standard error. (C) Reduced Brf in the fat body reduced adult weight compared with control adults. Error bars indicate standard error. (D–G) Silencing of brf specifically in the Drosophila fat body decreased peripheral insulin signalling. (D) brf silencing in the fat body abolished Akt phosphorylation at serine 505 in peripheral tissues while total Akt protein levels remained constant. Levels of β-tubulin were measured to ensure equal loading. (E) Decreasing Brf levels specifically in the fat body using the r4-GAL4 driver significantly increased dInR mRNA levels in peripheral tissues of these animals when compared with controls (P<0.05, Student's t-test; n=32 per genotype). All qRT–PCR error bars represent s.e.m. (F) There is an accumulation of dILP2 protein in the IPCs of r4>brf RNAi larval brains compared with controls (r4>+). (G) Quantified pixel intensities of DILP2 staining in IPC clusters in the larval brain (r4>+, n=12 and r4>brf RNAi, n=21; error bars represent standard deviation; P=000174). For (D–G), larvae were analysed at 96 h AEL.

Figure 3

The fat body-specific loss of Brf function phenocopies some aspects of the starvation response. Fat bodies were dissected from 72 h larvae and stained with Nile Red to visualize lipid droplets or lysotracker green to vizualize autophagosomes. (A–C) DIC and (D–F) Nile Red images of fat bodies isolated from control (r4>+) fed (A, D) and control 24 h starved larvae (B, E). (C, F) Fed larvae with fat body-specific reduction in Brf levels (r4>brf RNAi) are shown. (G–I) Lysotracker green images of fat bodies isolated from control fed (G) and r4>brf RNAi (I) larvae show no lysotracker staining. Starved control larvae exhibit a punctuate staining pattern (H, arrowheads) caused by the formation of autophagosomes. Images were all taken at the same exposure. Scale bars, 100 μm.

Figure 4

TOR signalling regulates Pol III-dependent transcription in Drosophila larvae. (A) Pol III-dependent transcripts were significantly decreased in wild-type (yw) larvae starved for dietary protein for 24 or 48 h compared with wild-type fed larvae. (B) tRNA levels were significantly decreased in tor^ΔP homozygous mutant larvae when compared with control (yw) larvae 48 h AEL (P<0.05, Student's t-test). (C) Pol III-dependent transcript levels were significantly decreased in larvae ubiquitously overexpressing tsc1/2 (da>tsc1/2) compared with controls (da>+) larvae 48 h AEL (P<0.05, Student's t-test). (D) Levels of Pol III-dependent transcripts were significantly reduced in S6K homozygous (dS6K^L1) mutant larvae when compared with control (yw) larvae 48 h AEL (P<0.05, Student's t-test). (E) Levels of Pol III-dependent transcripts were elevated in whole larvae following the ubiquitous expression of a tsc1 RNAi transgene by da-GAL4 (da>tsc1 RNAi) compared with controls (da>+, P<0.05, Student's t-test) at 72 h AEL. (F) Ubiquitous expression of a constitutively active form of S6K (da>S6K^TE1) significantly increased Pol III-dependent transcript levels in whole larvae compared with control (da>+) larvae 72 h AEL (P<0.05, Student's t-test). Each experiment was independently performed three times with n=32 per genotype. For each qRT–PCR error bars indicate s.e.m.

Figure 5

Brf is required for TOR-induced cell growth in both mitotically dividing and endoreplicating tissue in Drosophila larvae. (A–F) brf, tsc1 or tsc1,brf double mutant clones were induced in both wing discs (A–C) and fat body (D–G). Mutant clones, arrowheads; wild-type sister clones, arrows. Blue, DAPI staining; red, actin; green, GFP. (G) The areas of both mutant and wild-type cells in the fat body were measured and presented here as mean cell area compared with control. Genotypes: (A, D) hsflp¹²²; +; FRT82B, brf^EY02964/FRT82B, ubi-GFP; (B, E) hsflp¹²²; +; FRT82B, tsc1^Q87X/FRT82B, ubi-GFP; (C, F) hsflp¹²²; +; FRT82B, brf^EY02964, tsc1^Q87X/FRT82B, ubi-GFP. (A–C) Scale bar, 50 μm. (D–F) Scale bar, 100 μm.

Figure 6

Drosophila Maf1 is the regulatory link between TOR and Pol III activity. (A–F) qRT–PCR analyses of RNA extracted from whole larvae following the ubiquitous expression of a dMaf1 RNAi transgene (da>Maf1 RNAi) compared with control (da>+) larvae. TOR activity was modulated either by starving the larvae of dietary protein (A–C) or by feeding larvae rapamycin (D–F). (A–F) tRNA levels were significantly elevated following loss of dMaf1 (da>Maf1 RNAi) compared with controls (da>+) when TOR activity was high under normal fed or DMSO-treated conditions (P<0.05, Student's t-test). tRNA levels remain elevated in da>Maf1 RNAi animals even under starved or rapamycin-treated conditions when control tRNA levels are normally reduced (P<0.05, Student's t-test). For qRT–PCR error bars indicate s.e.m. (G) Immunoprecipitation of Brf and dMaf1 from Drosophila cultured S2 cell extracts revealed an enhanced association between Brf and dMaf1 following rapamycin treatment (compare lane 4 with 9 and lane 5 with 10).

Figure 7

dMyc activates Pol III-dependent transcription in vivo by two distinct mechanisms. (A) tRNA_i^Met, Brf and Trf mRNA levels were significantly decreased in dMyc homozygous mutants (dm⁴) compared with controls (1-14-2, P<0.05; Student's t-test) 48 h AEL. (B) tRNA synthesis and TFIIIB mRNA levels were significantly elevated in larvae following dMyc overexpression using the flp-out technique (hsflp/+;+;UAS-dMyc/act>CD2>Gal4) compared with controls (hsflp/+;+;act>CD2>Gal4/+, P<0.05; Student's t-test) at 120 h AEL following a 24-h induction in dMyc gene expression. (C) Immunoblotting of whole larval protein extracts with antibodies specific to Brf and β-tubulin revealed an increase in Brf protein levels following dMyc overexpression (using the flp-out technique) compared with controls at 120 h AEL following a 24-h induction in dMyc gene expression. (D) Co-immunoprecipitation studies revealed a specific association between Brf and dMyc proteins in wild-type (yw) Drosophila larval extracts 96 h AEL. (E) Fat body expression of dMyc (cg>UAS-dMyc) increases pupal volume compared with controls (cg>+). This effect is abolished when Brf levels are decreased in the larval fat body (cg>UAS-brf RNAi, UAS-dMyc) and pupae are similar in size to controls. cg>brf RNAi larvae fail to progress to the pupal stage. (F) Rapamycin treatment of dMyc mutants failed to decrease Pol III-dependent transcription further at 48 h AEL in whole larvae. (G, H) Overexpression of dMyc (using the flp-out technique) failed to reverse the decrease in tRNA synthesis in starved animals (G) and rapamycin-treated animals (H), as was measured by qRT–PCR of RNA extracted from whole larvae 72 h AEL. For all qRT–PCR experiments error bars indicate s.e.m.

Figure 8

A model for nutrient/TOR regulation of Pol III in Drosophila. Our data suggest the predominant mechanism by which nutrition/TOR controls Pol III is via Maf1 repression, since Maf1 inhibition completely reverses the decrease in tRNA synthesis caused by TOR inhibition. Myc is sufficient and necessary for Pol III transcription, through controlling levels of Pol III factors (such as Brf) and through interaction with Brf. TOR can control Myc protein levels (Parisi et al, 2011; Teleman et al, 2008—dashed arrow in model figure). But these effects probably do not play a major role in how TOR activates Pol III since our data show that—unlike Maf1 inhibition—maintaining Myc levels and activity cannot reverse the decrease in tRNA synthesis caused by TOR inhibition.