Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila
2012; Springer Nature; Volume: 31; Issue: 8 Linguagem: Inglês
10.1038/emboj.2012.33
ISSN1460-2075
AutoresLynne Marshall, Elizabeth J. Rideout, Savraj Grewal,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoArticle24 February 2012free access Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila Lynne Marshall Lynne Marshall Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Elizabeth J Rideout Elizabeth J Rideout Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Savraj S Grewal Corresponding Author Savraj S Grewal Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Lynne Marshall Lynne Marshall Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Elizabeth J Rideout Elizabeth J Rideout Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Savraj S Grewal Corresponding Author Savraj S Grewal Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada Search for more papers by this author Author Information Lynne Marshall1, Elizabeth J Rideout1 and Savraj S Grewal 1 1Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, Calgary, Alberta, Canada *Corresponding author. Department of Biochemistry and Molecular Biology, Clark H Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, HRIC, 3330 Hospital Drive, Calgary, Alberta, Canada T2N 4N1. Tel.: +1 403 210 6535; Fax: +1 403 210 9563; E-mail: [email protected] The EMBO Journal (2012)31:1916-1930https://doi.org/10.1038/emboj.2012.33 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction An important question in developmental biology concerns the mechanisms that control growth and final size in multicellular animals. Studies in different model organisms have identified many conserved cell–cell secreted factors and signalling pathways that control growth. One key regulator that has emerged from this work is the serine/threonine kinase, target-of-rapamycin (TOR; for reviews, see De Virgilio and Loewith, 2006; Wullschleger et al, 2006 and Foster and Fingar, 2010). From yeast to mammals, TOR activity is cell-autonomously stimulated by an array of extracellular cues such as amino acids, glucose and oxygen to control growth and proliferation (Arsham and Neufeld, 2006; Dann and Thomas, 2006; Wullschleger et al, 2006; Hietakangas and Cohen, 2009; Wang and Proud, 2009; Foster and Fingar, 2010). In addition, in metazoans TOR can be activated by an endocrine insulin/insulin-like growth factor (IGF) signalling pathway (Oldham and Hafen, 2003; Grewal, 2008; Teleman, 2009). Insulins and insulin-like peptides bind to receptors on the surface of target cells. Ligand-receptor binding then triggers a conserved intracellular signalling cascade involving phosphoinositol 3-kinase (PI3K) and Akt, ultimately leading to increased TOR activity (Bhaskar and Hay, 2007; Efeyan and Sabatini, 2009). While these cell–cell and intracellular signalling inputs to TOR are well defined, the key downstream outputs by which TOR mediates its effects on metabolism and growth in vivo are less clear. Considerable attention has focussed on the role of cellular protein synthesis as a regulator of cell growth. Extensive studies in mammalian cell culture have identified several mechanisms by which TOR can control mRNA translation (for reviews, see Proud, 2007; Ma and Blenis, 2009 and Sonenberg and Hinnebusch, 2009). For example, TOR can phosphorylate and inhibit the translational repressor eukaryotic initiation factor 4E-binding protein (4E-BP) leading to stimulation of protein synthesis (Thomas, 2002; Jastrzebski et al, 2007; Ma and Blenis, 2009). This translational mechanism is widely proposed as a key growth-regulatory target of TOR signalling (Dowling et al, 2010). These effects may not, however, account fully for the in vivo growth functions of TOR. For example, in Drosophila, TOR null mutants are lethal with severe growth defects (Oldham et al, 2000; Zhang et al, 2000) and overactivation of TOR signalling can promote considerable overgrowth; null mutants for 4E-BP, on the other hand, are viable with no effects on growth during development (Miron et al, 2001; Teleman et al, 2005). The regulation of ribosome synthesis is another TOR function important for protein synthesis and growth. Studies in yeast and mammalian cell culture have identified several mechanisms by which TOR can control the expression of ribosomal RNA (rRNA) and ribosome biogenesis genes (Mayer and Grummt, 2006). Moreover, recent work in Drosophila has emphasized the in vivo regulation of ribosome synthesis by TOR. For example, in larvae the insulin/TOR pathway controls the expression of ribosome synthesis genes via the transcription factors FOXO and Myc (Teleman et al, 2008; Li et al, 2010). In addition, the RNA polymerase I factor, TIF-IA, is required for rRNA synthesis and larval growth and is a downstream target of insulin/TOR signalling (Grewal et al, 2007). In this paper, we explore the regulation of RNA polymerase (Pol) III-dependent transcription as a growth-regulatory output of insulin/TOR signalling in Drosophila. Pol III is responsible for the synthesis of small non-coding RNAs that are essential for mRNA translation (e.g., 5S rRNA and transfer RNAs—tRNAs). Thus, control of Pol III may therefore represent another mechanism by which TOR alters protein synthesis to regulate growth. Studies on TOR signalling and Pol III have been exclusively limited to work in yeast and mammalian cell culture studies. For example, the multisubunit transcription factor TFIIIB is essential for Pol III transcription initiation, and nuclear extracts from either nutrient-deprived or TOR-inhibited yeast show reduced TFIIIB activity in vitro (Dieci et al, 1995; Sethy et al, 1995; Clarke et al, 1996; Zaragoza et al, 1998). Furthermore, in cultured mammalian cells the Brf (TFIIIB-related factor) subunit of TFIIIB is regulated downstream of several growth-regulatory signalling pathways including the TOR cascade (Goodfellow and White, 2007; Woiwode et al, 2008). These effects on TFIIIB/Pol III-dependent transcription in yeast and mammalian cells may reflect the ability of TOR to phosphorylate and inhibit the Pol III repressor Maf1, thus promoting transcription (Upadhya et al, 2002; Lee et al, 2009; Wei et al, 2009; Kantidakis et al, 2010; Michels et al, 2010; Shor et al, 2010). Mammalian Brf activity can also be stimulated by direct interaction with oncogenes such as c-Myc (White, 2005). While these in vitro studies have provided important molecular details about the regulation of Pol III in vitro, they do not address questions about metabolism, growth and size control in a developing multicellular animal: How does regulation of Pol III influence cell and tissue growth? Is Pol III required for the in vivo functions of TOR? If so, what are the regulatory mechanisms involved? Our approach has been to use Drosophila as a model system to examine the contribution of Pol III-dependent transcription to the control of cell and tissue growth in vivo. During Drosophila larval development, the period of the life cycle characterized by an immense increase in growth, the major function of TOR signalling is to couple dietary nutrition to cell and tissue growth (Britton et al, 2002). TOR activity is required to cell-autonomously control growth in all larval tissues. In addition, stimulation of TOR in specific tissues can also play a non-autonomous role in systemic growth. For example, in well-fed larvae, amino-acid import into fat cells activates TOR leading to relay of a signal to the brain to promote the release of several Drosophila insulin-like peptides (dILPs) from discrete neurosecretory cells (Ikeya et al, 2002; Geminard et al, 2009). These dILPs then circulate through the larval haemolymph and activate the insulin-signalling pathway to stimulate cell growth in all larval tissues. We show here that Brf is an essential effector of TOR in the control of both cell-autonomous and non-autonomous effects on growth and body size in Drosophila. Moreover, we present evidence for a prominent role for dMaf1, but only a limited role for Drosophila Myc (dMyc), in the control of Pol III by nutrient-TOR signalling in developing animals. Results Brf is required for both cellular and organismal growth in Drosophila larvae Brf, a conserved component of the TFIIIB complex, is limiting for Pol III-dependent transcription in yeast and mammals (Geiduschek and Kassavetis, 2001; Marshall et al, 2008). We therefore investigated if Brf is involved in controlling Pol III-dependent transcription and growth in Drosophila larvae. For these experiments, we analysed two publicly available lines (Bloomington Stock Center) carrying P-element insertions in the brf locus (brfEY02964 and brfc07161). Homozygous brfEY02964 flies were lethal and this lethality could be rescued by ubiquitous GAL4-dependent expression of a UAS-brf transgene. Homozygous brfEY02964 larvae also had reduced levels of both Brf protein (Figure 1A) and Pol III-dependent transcripts (Figure 1B) compared with control, wild-type larvae at the same developmental stage. Furthermore, levels of 7SL RNA were lower in brf mutants compared with controls; however, we did not detect any changes in the levels of 5S rRNA or the Pol I-dependent transcript, pre-rRNA (Supplementary Figure S1). Phenotypically, brfEY02964 larvae progressed through embryogenesis but arrested as second instar larvae, surviving for several days (Figure 1C). A similar phenotype was seen in flies transheterozygous for brfEY02964 and a deficiency that uncovers the brf locus (Df(3R)BSC565), suggesting that brfEY02964 is either a null or strong hypomorphic loss-of-function allele of brf. We therefore used this line as a brf mutant. Flies that were homozygous for the second P-element line, brfc07161, also exhibited lethality, but this could not be rescued by ubiquitous GAL4-dependent expression of a UAS-brf transgene. Hence, this P-element line must also be mutated in another essential gene, and so we did not study it any further. The growth inhibitory effects seen in homozygous brf mutant larvae could be phenocopied by expression of a UAS-brf RNAi construct using the ubiquitous daughterless (da)-GAL4 driver (Supplementary Figure S2). Reducing Brf protein levels in this manner also decreased rates of Pol III-dependent transcription (Supplementary Figure S2A and B) and reduced larval growth rates (Supplementary Figure S2C). Expression of brf RNAi in either the salivary gland (patched (ptc); Supplementary Figure S2D and E) or eye imaginal discs (eyeless (ey); data not shown) also led to a reduction in tissue growth. Importantly, the growth inhibitory effects of the brf RNAi transgene were reversed by overexpression of UAS-brf, indicating that the RNAi-mediated effects were specifically due to Brf knockdown (Supplementary Figure S2F and G). In contrast to the effects of Brf inhibition, we found that overexpression of Brf alone was not sufficient to stimulate Pol III activity or affect organismal growth (data not shown). Thus, Brf, probably through its role in driving Pol III-dependent transcription, is essential for both tissue and organismal growth in Drosophila larvae. Figure 1.Loss of Brf function leads to severe growth defects in Drosophila larvae. (A) Brf protein levels were reduced in brf mutant (brfEY02964) 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 brf RNAi) reduced larval growth rates and delayed pupation, with ∼15% of larvae failing to pupate and remaining as third instar larvae (Figure 2A). At wandering third instar stage, r4>brf RNAi larvae had significantly smaller wing imaginal discs than control larvae (Figure 2B). Subsequently, we found that r4>brf RNAi adults were smaller than controls and weighed less (Figure 2C). Similar but stronger effects were seen using another fat body driver (cg-GAL4); cg>brf RNAi larvae were significantly smaller than controls and failed to progress into the pupal stage (Supplementary Figure S4A). 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 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. Download figure Download PowerPoint We explored whether these organismal growth effects caused by inhibiting Brf in the fat body were a consequence of reduced systemic insulin signalling. To do so, we first examined phospho-Akt levels in the peripheral tissues of r4>brf RNAi animals by immunoblotting. Akt is a key downstream effector of the insulin pathway, and Akt activity can be measured by assaying for phosphorylation of a carboxy terminus serine residue at position 505. We found that phosphorylation of Akt at serine 505 was reduced in r4>brf RNAi larvae peripheral tissues (larval carcasses devoid of fat body), even though total Akt was still present at levels comparable to control animals (Figure 2D). Similarly, brf mutant larvae also had lower levels of Akt phosphorylation at serine 505 compared with age-matched wild-type whole larvae (Supplementary Figure S4B). To further confirm that the inhibition of larval growth caused by fat body silencing of Brf was due to reduced systemic insulin signalling, we measured the levels of dInR mRNA. Transcription of this gene is negatively regulated by the insulin/PI3K pathway through the activation of FOXO (Puig and Tjian, 2005), and hence levels of dInR mRNA act as an additional readout of insulin signalling (Delanoue et al, 2010). When we used r4-gal4 to drive brf RNAi in the fat body, we found an increase in dInR mRNA levels in peripheral tissues, consistent with a suppression of peripheral insulin signalling (Figure 2E). We saw a similar increase in dInR mRNA in both peripheral tissues from cg>brf RNAi (Supplementary Figure S4C), and also in brf mutant animals when compared with control animals (Supplementary Figure S4D). Finally, we examined whether these changes in systemic insulin signalling following the knockdown of Brf might be explained by either reduced expression or release of brain dILPs. Previous reports have shown that mRNA levels of dilp5, but not dilp2, are suppressed upon amino-acid starvation (Geminard et al, 2009). We saw no change in dILP mRNA expression levels in r4>brf RNAi larvae (Supplementary Figure S4E). In contrast, we saw reduced expression levels of the mRNAs encoding dilp2 and dilp5 in cg>brf RNAi larvae (Supplementary Figure S4F). We also found that dilp5 mRNA levels were reduced in brf mutants (Supplementary Figure S4G). Amino-acid deprivation also leads to reduced release of dILPs from the brain and hence dILP proteins are retained in the neurosecretory insulin producing cells (IPCs) of starved animals (Geminard et al, 2009). Using immunostaining, we also found that dILP2 protein was retained in the IPCs of brains from r4>brf RNAi larvae compared with controls (Figure 2F and G). Taken together, these data suggest that Brf function and hence Pol III-dependent transcription is required in the fat body to maintain normal systemic insulin signalling and growth. Given the organismal effects we observed following brf knockdown in fat cells, we examined whether Brf might be required for nutrient-dependent effects on fat body metabolism. To do so, we compared the fat bodies of starved larvae with those from larvae in which Brf had been specifically silenced in the fat body by expression of brf RNAi using the fat body driver r4-GAL4. Nutrient-deprivation/TOR inhibition induces marked changes in lipid metabolism (Colombani et al, 2003), which can be observed as an increase in lipid droplet size. Using both Differential Interference Contrast (DIC) microscopy and Nile Red staining, we observed an increased lipid droplet size in r4>brf RNAi larvae compared with control animals (Figure 3A, D versus C, F). These effects were similar to changes in lipid droplets in fat bodies dissected from either amino acid-deprived (Figure 3B and E) or tor mutant larvae (Zhang et al, 2000). Similar effects were seen when we expressed the UAS-brf RNAi transgene with another fat body driver, cg-GAL4 (Supplementary Figure S5A and B). Starvation for amino acids also stimulates a rapid induction of autophagy, a response that is required for organismal survival. We found that fat bodies from 4 h starved larvae showed a marked increase in autophagasomes by using lysotracker staining (Figure 3H). In contrast, we found that r4>brf RNAi fat bodies showed no induction of autophagy (Figure 3I), similarly to fat bodies dissected from fed larvae (Figure 3G). These results suggest that Brf and Pol III-dependent transcription in the Drosophila fat body are required for some but not all of the metabolic effects of nutrient availability. 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. Download figure Download PowerPoint Pol III transcription is stimulated by the TOR pathway The cell and organismal changes in metabolism, physiology and growth that we described for loss of Brf function are similar to those seen following the inhibition of TOR signalling. We therefore explored whether TOR regulates Pol III-dependent transcription in Drosophila, and whether this regulation is required for the in vivo functions of TOR signalling. To address this, we first measured levels of Pol III-dependent transcripts, by qRT–PCR, in Drosophila larvae following the modulation of the TOR pathway. Starvation for dietary protein leads to inhibition of TOR activity in larvae (Oldham et al, 2000; Zhang et al, 2000). We found that larvae starved in 20% sucrose/PBS had reduced levels of several Pol III-dependent transcripts such as the tRNAs, 5S rRNA and 7SL RNA (Figure 4A). To further investigate the involvement of the TOR pathway in Pol III regulation in vivo, we performed gene expression analyses in larvae in which we genetically manipulated TOR signalling. We first found that tor null mutants had significantly reduced levels of Pol III-dependent transcripts compared with age-matched control larvae, (Figure 4B). We also found that cultured Drosophila S2 cells treated with the TOR-specific inhibitor, rapamycin, also had reduced levels of Pol III-dependent products (Supplementary Figure S6). Similarly, we found that overexpression of negative regulators of the TOR pathway, TSC1/2 using the UAS-GAL4 system, resulted in a substantial reduction in Pol III-dependent transcript levels compared with control larvae (Figure 4C). Finally, we found levels of Pol III-dependent transcripts were reduced in homozygous mutants for S6 kinase, a key TOR effector (Figure 4D). We next asked if overactivation of the TOR pathway can increase Pol III-dependent transcription. To address this, we first ubiquitously expressed a tsc1 RNAi transgene using the da-GAL4 driver and found that these larvae had significantly increased levels of each of the Pol III-dependent transcripts measured (Figure 4E). We then examined larvae expressing a constitutively active form of the downstream TOR effector S6K, and found that levels of Pol III-dependent transcripts were significantly elevated in these larvae compared with controls (Figure 4F). Taken together, these data demonstrate that the TOR pathway is necessary and sufficient to stimulate Pol III-dependent transcription in developing larvae, in part through activation of S6 kinase. 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 torΔP homozygous mutant larvae when compared with control (yw) larvae 48 h AEL (P 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 (dS6KL1) mutant larvae when compared with control (yw) larvae 48 h AEL (P tsc1 RNAi) compared with controls (da>+, P S6KTE1) 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
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