InteractiveFly: GeneBrief

RNA polymerase III: Biological Overview | References

RNA polymerase III (RNAP III) synthesizes short, non-coding RNAs including tRNAs, the small 5S rRNA and the U6 snRNA. It comprises 17 subunits, ten of which form a structurally conserved catalytic core with additional subunits located on the periphery. Notably, five RNAP III subunits are shared among all three RNAPs and two others are shared between RNAPs I and III (Flybase).

Pharmacological therapies are promising interventions to slow down aging and reduce multimorbidity in the elderly. Studies in animal models are the first step toward translation of candidate molecules into human therapies, as they aim to elucidate the molecular pathways, cellular mechanisms, and tissue pathologies involved in the anti-aging effects. Trametinib, an allosteric inhibitor of MEK within the Ras/MAPK (Ras/Mitogen-Activated Protein Kinase) pathway and currently used as an anti-cancer treatment, emerged as a geroprotector candidate because it extended lifespan in the fruit fly Drosophila melanogaster. This study confirmed that Trametinib consistently and robustly extends female lifespan, and reduces intestinal stem cell (ISC) proliferation, tumor formation, tissue dysplasia, and barrier disruption in guts in aged flies. In contrast, pro-longevity effects of trametinib are weak and inconsistent in males, and it does not influence gut homeostasis. Inhibition of the Ras/MAPK pathway specifically in ISCs is sufficient to partially recapitulate the effects of trametinib. Moreover, in ISCs, trametinib decreases the activity of the RNA polymerase III (Pol III), a conserved enzyme synthesizing transfer RNAs and other short, non-coding RNAs, and whose inhibition also extends lifespan and reduces gut pathology. Finally, this study showed that the pro-longevity effect of trametinib in ISCs is partially mediated by Maf1, a repressor of Pol III, suggesting a life-limiting Ras/MAPK-Maf1-Pol III axis in these cells. The mechanism of action described in this work paves the way for further studies on the anti-aging effects of trametinib in mammals and shows its potential for clinical application in humans (Urena, 2024).

Average life expectancy in humans has doubled during the last 100 y, currently surpassing 83 y in wealthy countries such as Switzerland, Australia, and Japan. However, healthy lifespan, or "healthspan", is not increasing at the same rate. This has resulted in an increasing prevalence of age-related diseases, such as cardiovascular dysfunctions, cancers, and neurodegenerative disorders, associated with an escalating economic burden and pressure on healthcare services. Some pharmacological agents already used in the clinic, such as the mammalian target of rapamycin (mTOR)-inhibitor rapamycin, can counteract aging-related phenotypes and diseases in animal models. Repurposing of these and other drugs as potential geroprotective treatments is hence being proposed to compress the period of morbidity in older people. Evidence from animal models, including the tissues and pathologies improved by these drugs, the molecular pathways involved, and sexually dimorphic responses and side-effects are needed to accelerate the transition of these treatments to human clinical trials (Urena, 2024).

Trametinib is an anti-cancer drug currently used for the treatment of metastatic melanoma, anaplastic thyroid cancer, and non-small cell lung cancer and has been described as a potential anti-aging drug based on data from the fruit fly Drosophila melanogaster. It is an allosteric inhibitor of MEK, the Mitogen-Activated Protein Kinase (MAPK) of the Extracellular-Signal-Regulated Kinase (ERK), part of the Ras/MAPK pathway, a highly conserved signaling cascade of kinases controlling cell survival, proliferation, growth, and differentiation. The Ras/MAPK pathway can be activated by different receptor tyrosine kinases present at the plasma membrane of the cell, including insulin receptor and epidermal growth factor receptor. This results in the activation of Ras small nucleotide guanosine triphosphate hydrolases (Ras GTPases) and the downstream phosphorylation cascade composed of Raf, MEK, and ERK kinases. Once phosphorylated, ERK can modify a wide range of cytoplasmic and cytoskeletal protein substrates, as well as several nuclear transcription factors, and hence activate cell division, differentiation, survival, and growth (Urena, 2024).

Ras/MAPK pathway hyperactivation leads to uncontrolled cell proliferation and is one of the best-described mechanisms leading to tumor formation. On the other hand, direct inhibition of Ras orthologs and other components of the Ras/MAPK pathway extends lifespan in Drosophila and the budding yeast Saccharomyces cerevisiae, indirect inhibition of HRas extends lifespan in wild-type and tumor-free mice, and variants of HRAS1 in combination with APOE and LASS1 variants are associated with human longevity and healthy aging. Furthermore, pharmacological inhibition of the Ras/MAPK pathway with trametinib not only extends lifespan in Drosophila but also reduces cellular senescence in normal human dermal fibroblasts in vitro. Thus, emerging evidence suggests that Ras/MAPK pathway plays an essential role in the aging process and makes trametinib an interesting candidate geroprotector. However, little is known about the molecular and cellular processes that mediate this effect. (Urena, 2024).

Recently, RNA polymerase III (Pol III) has been described as a potential target for anti-aging treatments, as its inhibition in S. cerevisiae, the worm Caenorhabditis elegans, and Drosophila is sufficient to extend lifespan. Pol III is an evolutionarily conserved complex composed of 17 subunits and controls the transcription of different short untranslated RNAs, including transfer RNAs (tRNAs), which play an essential. Pol III has been shown to act downstream of TORC1, but whether its activity mediates the effects of other signaling pathways on aging remains unaddressed (Urena, 2024).

Drosophila was used to study the geroprotective effects of trametinib, in both females and males, and to analyze the molecular mechanisms at work. Trametinib consistently extends lifespan and ameliorates aging-related gut pathology in females, while in males it has minor effects on lifespan and no detectable impact on gut health. Further, in females, trametinib was shown to reduces Pol III activity in intestinal stem cells (ISCs) and that the full life-extending effect of trametinib requires Pol III inhibitor Maf 1 in ISCs. These findings show that the inhibitory effect of trametinib on Pol III activity in ISCs mediates, at least partially, its pro-longevity effect and the reduction of gut pathology in aging females (Urena, 2024).

Trametinib is a Food and Drug Administration (FDA)-approved anticancer drug with the potential to be repurposed as a geroprotector. This study now showns that it reduces age-related ISC hyperproliferation in both sexes, decreases epithelial dysplasia and tumor formation, and maintains gut integrity in females. The Ras/MAPK pathway controls cell proliferation in multiple tissues across organisms. In Drosophila guts, it plays a central role in preserving gut homeostasis, as it is necessary for maintaining ISC proliferation both in unchallenged conditions and under stress in young flies, where reduced activity of Ras/MAPK signaling leads to reduced ISC proliferation, while activation triggers high proliferation levels. The results extend previous studies in young flies and show that Ras/MAPK signaling is necessary for age-related hyperproliferation leading to dysplasia and tumor formation in old flies, contributing to organismal aging (Urena, 2024).

Trametinib treatment or genetic inhibition of Ras/MAPK pathway in ISCs also reduced the disruption of the gut barrier in old flies. The deterioration of the intestinal barrier with age is a conserved pathology among different animal models including worms, fish, mice, and monkeys, and some markers of intestinal disruption have been observed in elderly humans. In Drosophila, loss of intestinal barrier stability correlates with gut dysbiosis, including increased bacteria in the gut, changes in microbiota composition and systemic inflammation. Similarly, microbial spread and systemic inflammation following intestinal dysfunction have been observed in aged mice and vervet monkeys. Thus, impaired intestinal function is closely related to health decline and disease in aged organisms, supporting further studies to confirm the effect of trametinib on intestinal homeostasis in higher animals (Urena, 2024).

The increase in ISC proliferation in old flies is significantly lower in males than in females, leading to a lower rate of dysplasia and tumor formation and contributing to healthier guts at old ages with a better maintenance of barrier function. Although trametinib significantly reduced ISC proliferation in males, no significant effect of the treatment was detected on intestinal dysplasia, tumor formation, or barrier function. Moreover, the effect of trametinib on male lifespan, although significant in a minority of trials, was much weaker. Increasing the concentration of the drug in the food and measurements of Ras/MAPK pathway activity in the gut indicated that the much lower effects of trametinib on gut pathology and lifespan in males could not be solely attributed to their lowered food consumption relative to females. The more likely explanation, and one consistent with previous observations, is that trametinib increases survival in part by reducing gut pathology, which is limiting for lifespan in females but not in males. In those trials where male lifespan was extended, it is possible that some micro-environmental condition (e.g., microbes) induced some level of pathology in males that was rescued by the drug. Loss of intestinal homeostasis and barrier dysfunction is common in both sexes in mammals, contributing to the onset of aging-related inflammatory and metabolic disorders. Thus, the effect of trametinib on gut health should be investigated in mammalian models, as it could be beneficial in both sexes (Urena, 2024).

Pol III, an RNA polymerase essential for protein translation and cell growth, limits lifespan in yeast, worms, and flies, and reducing its activity specifically in ISCs extends lifespan and ameliorates gut pathology in old female flies. The Ras/MAPK pathway controls growth and proliferation promoting Pol III activity and tRNA synthesis through phosphorylation of the Pol III repressor Maf1 in the fruit fly (Sriskanthadevan-Pirahas, 2018). This inhibitory mechanism is conserved in mammals to promote protein synthesis, cell proliferation, and tissue growth. This study has shown that trametinib decreases Pol III activity in ISCs, and our results suggest that Pol III acts downstream of the Ras/MAPK in ISCs to limit survival. Moreover, the life-extending effect of trametinib was found to partially depend on Maf1 expression in ISCs. Thus, a model is proposed in which the inhibition of MEK in ISCs decreases pERK signaling, allowing unphosphorylated Maf1 to bind and inhibit Pol III in the nucleus, preventing its transcriptional activity. This contributes to extending lifespan and ameliorating the age-associated gut pathology. (Urena, 2024).

Several studies have shown that mTORC1 directly phosphorylates and inactivates Maf1 to stimulate Pol III activity and tRNA synthesis. Moreover, Pol III exerts a role on the pro-longevity effect of rapamycin, mTORC1 inhibitor, in Drosophila. This means Ras/MAPK signaling is not the only pathway to regulate Maf1 and tRNA synthesis in ISCs, and rapamycin and trametinib probably share this mechanism of action to extend lifespan. However, the fact that the pro-longevity effects of rapamycin and trametinib are almost completely additive suggests that other mechanisms of their action exist and that they may be complementary (Urena, 2024).

As well as consistently extending lifespan in Drosophila females, trametinib reduces translational errors in vitro in S2R+ cells, decreases insulin resistance in obese wild type and genetically obese mice, diminishes the proportion of senescent cells in senescent human dermal fibroblast cultures, and increases autophagy in pancreatic ductal adenocarcinoma cells, among others. Furthermore, its anti-inflammatory effects have been widely described in different diseases including cancers, cystic fibrosis, acute lung injury, or traumatic brain injury. The effect of trametinib on Pol III activity in ISCs and gut pathology described in this work adds another piece of evidence to its anti-aging effect, paving the way for further analysis in higher animal models while presenting trametinib as a solid candidate for future geroprotective treatments. Meanwhile, further experiments will be necessary to fully understand the impact of trametinib in all tissues and pathologies, as well as the molecular mechanisms responsible. This knowledge would be greatly beneficial to advance toward the potential repurposing of trametinib as a new anti-aging therapy (Urena, 2024).

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

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. This study reports on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. Activation of Ras/ERK signalling was found to promote tRNA synthesis both in vivo and in cultured Drosophila S2 cells. Pol III function was shown to be required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. The transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead, Ras signalling was shown to promote Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. It is proposed 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 (Sriskanthadevan-Pirahas, 2018).

RNA polymerase III limits longevity downstream of TORC1

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. This study shows that Pol III limits lifespan downstream of TORC1. A reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target (Filer, 2017).

The task of carrying out transcription in the eukaryotic nucleus is divided among RNA Pol I, II and III. This specialization is evident in the biogenesis of the translation machinery, a task that requires the co-ordinated activity of all three polymerases: Pol I generates the 45S pre-rRNA that is subsequently processed into mature rRNAs, Pol II transcribes various RNAs including mRNAs encoding ribosomal proteins, while Pol III provides the tRNAs and 5S rRNA. This costly process of generating protein synthetic capacity is tightly regulated to match the extrinsic conditions and the intrinsic need for protein synthesis by the key driver of cellular anabolism, TORC1. The central position of TORC1 in the control of fundamental cellular processes is mirrored by the notable effect of its activity on organismal physiology: following its initial discovery in worms, inhibition of TORC1 has been demonstrated to extend lifespan in all tested organisms, from yeast to mice, with beneficial effects on a range of age-related diseases and dysfunctions. TORC1 strongly activates Pol III transcription and this relationship suggests the possibility that inhibition of Pol III promotes longevity (Filer, 2017).

In Saccharomyces cerevisiae, each of the 17 Pol III subunits is encoded by an essential gene. This study generated a yeast strain in which the largest Pol III subunit (C160, encoded by RPC160, also known as RPO31) is fused to the auxin-inducible degron (AID). The fusion protein can be targeted for degradation by the ectopically expressed E3 ubiquitin ligase (OsTir) in the presence of indole-3-acetic acid (IAA) to achieve conditional inhibition of Pol III. It was confirmed that IAA treatment triggered degradation of the fusion protein, and it was observed that IAA treatment also improved the survival of the RPC160-AID strain upon prolonged culture. In addition, IAA treatment of the control strain lacking the AID fusion reduced its survival relative to both the same strain in the absence of IAA and to the RPC160-AID strain in the presence of IAA. Hence, Pol III depletion appears to extend the chronological lifespan in yeast. While IAA had no substantial effect on the survival of a strain carrying the AID domain fused to the largest subunit of Pol II (RPB220, also known as RP021), this strain appeared to survive better than the control strain did in the presence of IAA, indicating that inhibition of Pol II may also extend chronological lifespan. Chronological lifespan of yeast is a measure of survival in a nutritionally limited, quiescent population, whereas replicative lifespan measures the number of daughters produced by a single mother cell in its lifetime. No evidence was found that inhibition of Pol III causes an increase in the replicative lifespan in yeast (Filer, 2017).

The observed increase in chronological lifespan may simply indicate increased stress resistance and hence be of limited relevance to organismal ageing. To examine the role of Pol III in organismal ageing directly, animal models were examined. RNA-mediated interference (RNAi) was initiated against rpc-1, the Caenorhabditis elegans orthologue of RPC160, in worms from the L4 stage, causing a partial knockdown of rpc-1 mRNA. This consistently extended the lifespan of worms at both 20°C and 25°C. To reduce Pol III activity in Drosophila melanogaster, a P-element insertion that deletes the transcriptional start site of the gene encoding the Pol III-specific subunit C53 (CG5147^EY22749, henceforth called dC53^EY, was backcrossed into a healthy, outbred population of flies. Homozygous dC53^EY/EY mutants were not viable, but heterozygous females had reduced dC53 mRNA levels and lived longer than controls. Taken together, these data strongly indicate that Pol III limits lifespan in multiple model organisms and conversely, that partial inhibition of its activity is an intervention that increases longevity in multiple species (Filer, 2017).

The longevity of an animal can be governed from a single organ. In the worm, this role is often played by the gut. To restrict the rpc-1 knockdown to the gut, worms were used that were deficient in rde-1, in which the RNAi machinery deficiency is restored in the gut by gut-specific rde-1 rescue. rpc-1 RNAi extended the lifespan of this strain, both at 20°C and 25°C. Similarly, in the adult fly, driving an RNAi construct targeting the RPC160 orthologue (CG17209, henceforth called dC160,with the mid-gut-specific, RU486-inducible driver TIGS extended the lifespan of females, while the presence of the inducer (RU486) did not affect survival of the control strains. The longevity phenotype could also be recapitulated with RNAi against dC53, another Pol III subunit, indicating that the phenotype was not subunit-specific or due to off-target effects. As well as the gut, longevity can also be associated with the fat body and neurons in flies. However, the longevity phenotype caused by dC160 RNAi appears to be specific to the gut, since no significant lifespan extension was observed upon induction of dC160 RNAi in the fat body of the adult fly, and only a modest, albeit significant, extension resulted from neuronal induction of dC160 RNAi (Filer, 2017).

The worm gut is composed of only post-mitotic cells. In flies, as in mammals, the adult gut epithelium contains mitotically active intestinal stem cells, and the mid-gut-specific driver TIGS appears to be active in at least some ISCs, prompting restricting of dC160 RNAi induction to this cell type. ISC-specific dC160 RNAi, achieved with the GS5961 driver, was sufficient to promote longevity. In summary, Pol III activity in the gut limits survival in worms and flies, and in the fly, Pol III can drive ageing specifically from the gut stem-cell compartment (Filer, 2017).

The consequences of Pol III inhibition in the fly gut was assessed. Pol III acts to generate precursor tRNAs (pre-tRNAs) that are processed rapidly to mature tRNAs. Owing to their short half-lives, pre-tRNAs are useful as readouts of in vivo Pol III activity. Profiling the levels of specific pre-tRNAs, pre-tRNA^His, pre-tRNA^Ala and pre-tRNA^Leu, relative to the levels of U3 (a small nucleolar RNA transcribed by Pol II) revealed a moderate but significant reduction in Pol III activity upon gut-specific induction of dC160 RNAi. The three polymerases can be directly coordinated to generate the translation machinery. Indeed, Pol III inhibition had knock-on effects on Pol I- but not Pol II-generated transcripts, revealing partial cross-talk. dC160 RNAi also reduced protein synthesis in the gut, consistent with reduced Pol III activity. These effects (reduction in pre-tRNAs or protein synthesis) were not observed after feeding RU486 to the driver-only control. The reduction in protein synthesis was not pathological: total protein content of the gut was unaltered; fecundity, a sensitive readout of a female's nutritional status, was unaffected; and the flies' weight, triacylglycerol and protein levels remained unchanged. Reduced protein synthesis can liberate protein-folding machinery from protein production and increase homeostatic capacity. Indeed, induction of dC160 RNAi in the gut increased the resistance of adult flies to proteostatic challenge with tunicamycin for TIGS-only control. Hence, Pol III can fine-tune the rate of protein synthesis in the adult fly gut without obvious detrimental outcomes, while increasing resistance to proteotoxic stress (Filer, 2017).

Having demonstrated the relevance of Pol III for ageing, whether it acts on lifespan downstream of TORC1 was investigated in Drosophila. Numerous observations in several organisms support the model in which TORC1 localizes on Pol III-transcribed loci and promotes phosphorylation of the components of the Pol III transcriptional machinery to activate transcription, in part by inhibition of the Pol III repressor, Maf1. Using chromatin immunoprecipitation (ChIP) with two independently generated antibodies against Drosophila TOR (target of rapamycin), TOR enrichment was observed on Pol III-target genes in the adult fly, relative to Pol II targets. Inhibition of TORC1 by feeding rapamycin to flies reduced the levels of pre-tRNAs in whole flies. Rapamycin also reduced pre-tRNA levels specifically in the gut relative to U3. Since rapamycin results in re-scaling of the gut, evidenced by the reduction in the total RNA content of the organ, it was also confirmed that the drug reduced pre-tRNA levels relative to total RNA. Interestingly, rapamycin did not cause a decrease in 45S pre-rRNA in the gut, suggesting a lack of sustained Pol I inhibition. Additionally, gut-specific overexpression of Maf1 reduced the levels of pre-tRNAs and extended lifespan, confirming that Maf1 acts on Pol III in the adult gut. These data are consistent with TORC1 driving systemic and gut-specific Pol III activity in the adult fruitfly (Filer, 2017).

To examine whether the lifespan effects of Pol III are downstream of TORC1, adult-onset Pol III inhibition was combined with rapamycin treatment. Rapamycin feeding or gut-specific dC160 RNAi resulted in the same magnitude of lifespan extension. The two treatments were not additive, consistent with their acting on the same longevity pathway. The same effect was observed with RNAi against dC53 in the gut, as well as when dC160 RNAi was restricted to the ISCs. Importantly, rapamycin feeding also inhibited phosphorylation of the TORC1 substrate, S6 kinase (S6K), in both the gut and the whole fly, and decreased fecundity, while gut-specific C160 RNAi did not have these effects. This confirms that Pol III inhibition does not impact TORC1 activity locally or systemically, and therefore, Pol III acts downstream of TORC1 in ageing (Filer, 2017).

TORC1 inhibition is known to ameliorate age-related pathology and functional decline of the gut. Whether inhibition of Pol III was sufficient to block the dysplasia resulting from hyperproliferation and aberrant differentiation of ISCs was examined by assessing the characteristic, age-dependent increase in dividing phospho-histone H3 (pH3)-positive cells. Inducing dC160 RNAi in the fly gut or solely in the ISCs ameliorated this pathology. These treatments also counteracted the age-related loss of gut barrier function, decreasing the number of flies displaying extra-intestinal accumulation of a blue food dye (the 'Smurf' phenotype). It as also found that rpc-1 RNAi reduced the severity of age-related loss of gut-barrier function in worms. In Drosophila, gut health and TORC1 inhibition are specifically linked to female survival. Indeed, induction of dC160 RNAi in the gut had a sexually dimorphic effect on lifespan, as the effect on males, although significant, was lower in magnitude relative to the effect on females. Overall, the data show that gut- or ISC-specific inhibition of Pol III, which extends lifespan, is sufficient to ameliorate age-related impairments in gut health, which may be causative of or correlate with this longevity (Filer, 2017).

This study demonstrates that the adult-onset decrease in the growth-promoting anabolic function mediated by Pol III in the gut, and specifically in the intestinal stem-cell compartment, is sufficient to recapitulate the longevity benefits of rapamycin treatment. Pol III activity is essential for growth; its detrimental effects on ageing suggest an antagonistic pleiotropy in which wild-type levels of Pol III activity are optimised for growth and reproductive fitness in early life but prove detrimental for later health. This study reveals a fundamental role for Pol III in adult physiology, implicating wild-type Pol III activity in age-related stem-cell dysfunction, declining gut health and organismal survival downstream of nutrient signalling pathways. The longevity resulting from partial Pol III inhibition in adulthood is likely to result from the reduced provision of protein synthetic machinery; however, differential regulation of tRNA genes or Pol III-mediated changes to chromatin organization may also be involved, as has been suggested in other contexts (Arimbasseri, 2016). The strong structural and functional conservation of Pol III in eukaryotes suggests that studies of its influence on mammalian ageing are warranted and could lead to important therapies (Filer, 2017).

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

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. This study shows that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. TOR activity was shown to control Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, loss of the Pol III factor, Brf, was shown to lead 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, this study found 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 (Marshall, 2012).

This study found that 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 reducing TOR activity. These findings extend those observed in both yeast and mammalian cell culture, and suggest an important role for dMaf1 in vivo in developing tissues. The exact mechanism by which Maf1 functions is not clear, but it may involve inhibition of Brf and Pol III recruitment to genes, possibly by direct binding or association with Brf/Pol III. Indeed, an enhanced association was seen between dMaf1 and Brf1 upon TOR inhibition. The role of dMyc was explored as a potential link between nutrient-TOR signalling and Pol III. dMyc was found to be both necessary and sufficient for the control of Pol III activity during development. As previously reported in both mammalian and Drosophila culture, it was possible to identify an interaction between dMyc and Brf (Gomez-Roman, 2003; Steiger, 2008). In addition, a role has been identified for dMyc in controlling the levels of components of the Pol III machinery, including both Trf and Brf which form part of the TFIIIB complex. Thus, dMyc likely has both direct and indirect effects on Pol III activity in Drosophila. These effects are necessary for both dMyc-induced cell growth (Steiger, 2008) and, as is shown in this study, for the non-autonomous increases in body size caused by dMyc in fat cells. Previous studies have shown that, in Drosophila, TOR controls Myc protein levels. But these effects on Myc probably do not play major role in how TOR activates Pol III since the data show that, unlike inhibition of Maf1, maintaining Myc levels and activity cannot reverse the decrease in tRNA synthesis caused by TOR inhibition. Moreover, if Myc protein levels were limiting for TOR-dependent control of Pol III, then it would not be expected that knockdown of Maf1 could completely reverse the effects of rapamycin/starvation. Given that Maf1 inhibition did not influence levels of Pol III factors, pre-rRNA or RP gene mRNA—transcripts that are upregulated by dMyc—it is unlikely that Maf1 influences Myc function. It was found that rapamycin feeding could not exacerbate the reduction of tRNA levels seen in dMyc null mutants. This result in principle may suggest that TOR signalling does not exert any dMyc-independent effects on Pol III function. But, it is suggested that this finding probably occurs because in the absence of Myc, Pol III activity may be approaching basal levels and cannot be significantly decreased much further. Taken together, although these data may not completely rule out some contribution of Myc to TOR-dependent control of Pol III, they do indicate that it is not the major contributor (Marshall, 2012).

It is clear that both TOR and Myc are essential regulators of Pol III. But, it is likely that while TOR can control Myc levels, both TOR and Myc can also function in parallel and independently of each other. Previous studies have shown that overactivation of TOR signalling could not promote growth when Myc was inhibited, but at the same time Myc overexpression could not promote growth when TOR was inhibited. These findings and the current data suggest that TOR and Myc cannot necessarily be placed in a simple, linear pathway. Recent studies in Drosophila have emphasized how other conserved growth-regulatory pathways, particularly those that control growth of the imaginal tissues (such as Wingless, EGF/Ras, the Hippo-Yorkie pathway and Bantam RNAi) function via control of dMyc. Thus, dMyc may play a role in coupling these pathways to the control of Pol III activity to stimulate cell growth and proliferation (Marshall, 2012).

It is interesting to speculate as to which Pol III targets are important for growth control. Pol III regulates the expression of several short non-coding RNAs, such as the tRNAs, 5S rRNA and 7SL RNA. Regulation of 5S rRNA production by Brf could influence ribosome synthesis and hence growth. However, it was found that loss of Brf did not inhibit Pol I activity or alter levels of rRNA, suggesting that Brf probably does not directly influence ribosome numbers. One attractive possibility is that levels of the tRNAs may be limiting for translation and growth. In support of this notion, a recent paper showed that overexpression of Brf increased tRNA levels and promoted proliferation and transformation of cultured mammalian fibroblasts (Marshall, 2008). These effects of Brf were phenocopied by just increasing levels of tRNA_i^Met, and were associated with augmented mRNA translation and increased protein levels of growth promoters such as c-Myc and cyclin D1. No consistent increase was seen in tRNAs when Brf was overexpressed in larvae, perhaps because levels of other components of the TFIIIB complex are limiting in flies. Nevertheless, by controlling Brf activity and tRNA synthesis, TOR could promote translation of growth regulators and drive larval growth. In fact, a recent paper indicated that TOR signalling in Drosophila regulates dMyc protein levels, but not dMyc mRNA levels, consistent with a possible role for translational control (Marshall, 2012).

One interesting result of this work was the identification of a non-cell autonomous role for Brf in organismal growth. Specifically, it was found that Brf activity in the fat cells of Drosophila larvae could influence larval growth and final size. A role for TOR in the fat body has been shown to exist as a relay to control peripheral insulin signalling. In feeding larvae, amino-acid input into fat cells activates TOR, leading to transmission of a secreted signal from fat to brain to increase dILP expression and release from brain IPCs. These data suggest that stimulation of Pol III activity may be an important downstream effector of this adipose function of TOR. Thus, adipose-specific silencing of Brf led to reduced peripheral insulin signalling, slower larval growth rate and reduced final body size. As in starved larvae, this study found that loss of brf led to reduced expression of dilp mRNA (seen in both brf mutants and cg>brf RNAi larvae) and reduced dILP release from the brain. Moreover, given that levels of phospho-Akt are lower, and levels of dInR (a FOXO target) are higher in tissues from both brf mutant and r4>brf RNAi larvae it is clear that systemic insulin signalling is reduced when Brf is inhibited in the fat body. This study also found that another fat phenotype associated with starvation and loss of TOR, accumulation of lipid droplets, was phenocopied by loss of Brf. However, the autophagy phenotype of starved larval fat bodies was not phenocopied by loss of Brf. Therefore, Brf and Pol III function in the Drosophila fat body may mediate some, but not all of TOR's effects on growth and metabolism. The exact nature of the fat-to-brain secreted factor that controls insulin release in flies is not yet known, but perhaps translation of this signal, if it is a peptide or secreted protein, is influenced by changes in tRNA synthesis and translation rates. Indeed, it has been shown that dMyc activity in the fat body was also important for controlling systemic insulin signalling, growth and body size. This effect of dMyc correlated with elevated expression of ribosome biogenesis genes and increased nucleolar size, an index of ribosome synthesis. dMyc overexpression can also stimulate Pol III and tRNA levels, and the increase in body size caused by fat body overexpression of dMyc is reversed by knockdown of Brf. These data suggest that regulation of mRNA translational capacity is a key step downstream of TOR and dMyc in fat cells to control signalling to IPCs (Marshall, 2012).

Together, these data suggest that mRNA translational control may underlie a role for the fat body as an endocrine organ. A similar theme is emerging in mouse models. Mammalian adipose tissue is known to secrete adipokines and leptin to influence organismal metabolism and growth. The secretion of many of these factors is influenced by diet, suggesting a regulatory role for TOR signalling. Genetic inhibition of either TOR and S6K in mice leads to alterations in metabolic activity in adipose tissue. Moreover, loss of the translational repressors, 4E-BP1 and 4E-BP2, both of which are downstream TOR effectors, alters lipid and glucose metabolism in mice. To date, there are no mouse models of Pol III. However, it is interesting to speculate that changes in Pol III and tRNA synthesis are involved in mediating effects of TOR in adipose tissue in mice. Regulation of Pol III by TOR may also be important in the metabolic control of other processes. For example, TOR is a conserved regulator of organismal stress responses and lifespan. These stress responses rely on TOR's ability to control translation. It is suggested that regulation of Pol III and tRNA synthesis may also be a mode of control. Further organismal studies, using genetic modulation of Pol III function, should provide additional insights into these points (Marshall, 2012).

The TOR kinase is one of the best-established growth regulators. In virtually all animals, TOR activity can be stimulated by extracellular cues such as growth factors, nutrients and oxygen to control cell, tissue and organismal growth (Marshall, 2012).

Despite the knowledge of the signalling inputs to TOR, little is known about the mechanisms that allow TOR to modulate cell metabolism and drive growth. Most studies on metabolic functions modulated by TOR have been confined to yeast and mammalian cell culture. These studies have been important in defining roles for TOR in protein synthesis, nutrient uptake and metabolism and autophagy. But they leave open the question of what mechanisms operate in vivo to control tissue and organ growth during animal development. Genetic studies in Drosophila have been pivotal in this regard. This study shows that the ability of the TOR pathway to control transcription through Pol III governs cell, tissue and ultimately organismal growth in Drosophila. Given that Pol III drives transcription of several non-coding RNAs required for mRNA translation, it is suggested that the stimulation of Pol III by TOR enhances the protein synthetic capacity of cells. Previous study have shown that Drosophila TOR also controls synthesis of rRNA synthesis, via the RNA polymerase I factor, TIF-IA (Grewal, 2007). Moreover, recent studies in Drosophila larvae demonstrated that the insulin/TOR pathway regulates the expression of ribosome biogenesis genes via the transcription factors FOXO and Myc. Thus, in Drosophila, tissue and organismal growth relies on the ability of TOR to regulate all three nuclear RNA polymerases to ultimately promote protein synthesis. Given that regulation of all three polymerases is a conserved function for TOR, it is suggested that these mechanisms may also underlie tissue and organ growth in mammalian development (Marshall, 2012).

The Pol III transcription factor Brf has been shown to be an essential component of the TFIIIB complex responsible for recruiting Pol III to gene promoters. This work indicates that Brf activity is required for Drosophila development. Patterning and cell fate specification appear normal in brf embryos. However, once these mutants hatch as larvae they fail to grow. The data suggest that this growth arrest phenotype reflects a role for Brf activity downstream of TOR. Brf was found to be cell-autonomously required for growth in both endoreplicating cells, which make up the bulk of larval mass, and the mitotically dividing cells of the imaginal discs. In particular, brf mutant wing disc cell clones were found to be outcompeted by wild-type neighbours. This cell competition phenotype is seen in mutants for other genes required for protein synthesis, such as the ribosomal proteins and Myc. An important finding was that the overgrowth caused by loss of TSC1 (and hence increased TOR activity) was blocked in brf mutant cells. In mammalian cells, Brf activity is induced by cues that promote cell growth (e.g., during hypertrophic growth of cardiac cells) whereas cell differentiation leads to inhibition of Brf. In fact, overexpression of Brf alone can promote proliferation and transformation in immortalized fibroblasts. Mutations in tumour suppressors such as TSC are common in cancer and lead to elevated TOR activity and promotion of tumour growth. Based on the current data, it is suggested that Brf is required in vivo for both normal tissue growth and TOR-induced tumour growth (Marshall, 2012).

Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling

The target-of-rapamycin pathway couples nutrient availability with tissue and organismal growth in metazoans. The key effectors underlying this growth are, however, unclear. This study shows that Maf1, a repressor of RNA polymerase III-dependent tRNA transcription, is an important mediator of nutrient-dependent growth in Drosophila. Nutrients were found to promote tRNA synthesis during larval development by inhibiting Maf1. Genetic inhibition of Maf1 accelerates development and increases body size. These phenotypes are due to a non-cell-autonomous effect of Maf1 inhibition in the fat body, the main larval endocrine organ. Inhibiting Maf1 in the fat body increases growth by promoting the expression of brain-derived insulin-like peptides and consequently enhanced systemic insulin signaling. Remarkably, the effects of Maf1 inhibition are reproduced in flies carrying one extra copy of the initiator methionine tRNA, tRNA(i)(Met). These findings suggest the stimulation of tRNA(i)(Met) synthesis via inhibition of dMaf1 is limiting for nutrition-dependent growth during development (Rideout, 2012).

Functions of RNA polymerase III orthologs in other species

Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. This study conducted mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). This site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, this study has uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription (Shekhar, 2023).

Contrasting effects of whole-body and hepatocyte-specific deletion of the RNA polymerase III repressor Maf1 in the mouse

MAF1 is a nutrient-sensitive, TORC1-regulated repressor of RNA polymerase III (Pol III). MAF1 downregulation leads to increased lipogenesis in Drosophila melanogaster, Caenorhabditis elegans, and mice. However, Maf1 (-/-) mice are lean as increased lipogenesis is counterbalanced by futile pre-tRNA synthesis and degradation, resulting in increased energy expenditure. Chow-fed Maf1 (-/-) mice were compared with Chow- or High Fat (HF)-fed Maf1 (hep-/-) mice that lack MAF1 specifically in hepatocytes. Unlike Maf1 (-/-) mice, Maf1 (hep-/-) mice become heavier and fattier than control mice with old age and much earlier under a HF diet. Liver ChIPseq, RNAseq and proteomics analyses indicate increased Pol III occupancy at Pol III genes, very few differences in mRNA accumulation, and protein accumulation changes consistent with increased lipogenesis. Futile pre-tRNA synthesis and degradation in the liver, as likely occurs in Maf1 (hep-/-) mice, thus seems insufficient to counteract increased lipogenesis. Indeed, RNAseq and metabolite profiling indicate that liver phenotypes of Maf1 (-/-) mice are strongly influenced by systemic inter-organ communication. Among common changes in the three phenotypically distinct cohorts, Angiogenin downregulation is likely linked to increased Pol III occupancy of tRNA genes in the Angiogenin promoter (Willemin, 2023).

mTORC1 directly phosphorylates and regulates human MAF1

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. This study found that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. These results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1 (Michels, 2010).

Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes

Mouse myeloma cells have previously been shown to contain two chromatographically distinct forms of RNA polymerase III (designated III(A) and III(B)). The enzymes are unaffected by low alpha-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for III(A) and III(B)) at higher toxin concentrations. RNA polymerase I was unaffected at all alpha-amanitin concentrations tested. Myeloma RNA polymerases II, III(A), and III(B) appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic alpha-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases. Isolated myeloma nuclei and nucleoli continue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by alpha-amanitin, but only at high concentrations; and the alpha-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases III(A) and III(B). In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by alpha-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high alpha-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems. These results unequivocally demonstrate the role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species (Weinmann, 1974a).

Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection

DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.02 mug/ml), RNA polymerase III was completely sensitive to high concentrations of alpha-amanitin (50% inhibition at 20 mug/ml) and RNA polymerase I was totally resistant to concentrations of alpha-amanitin less than or equal to 200 mug/ml. RNA synthesis by the endogenous RNA polymerase activities in nuclei isolated from infected cells was completely sensitive to alpha-amanitin, thus suggesting that RNA polymerase I is not involved in viral DNA transcription even though it is present in these cells. The alpha-amanitin inhibition curve was biphasic and showed inflection points at about 0.02 and 20 mug/ml, suggesting the participation of both RNA polymerases II and III in the synthesis of RNA in these nuclei. Furthermore, at least a large fraction of the synthesis of the nuclear precursors to viral mRNA, monitored by hybridization to viral DNA, showed the same sensitivity to alpha-amanitin as did RNA polymerase II; and the synthesis of both viral 5.5S RNA and (presumably cellular) 5S RNA in the isolated nuclei exhibited the same sensitivity to alpha-amanitin as did purified RNA polymerase III. Thus, these data provide strong supporting evidence for previous studies which suggested the involvement of an RNA polymerase II in transcription of the adenovirus genome and demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA (Weinmann,, 1974b).

Search PubMed for articles about Drosophila RNA polymerase III

Filer, D., Thompson, M. A., Takhaveev, V., Dobson, A. J., Kotronaki, I., Green, J. W. M., Heinemann, M., Tullet, J. M. A., Alic, N. (2017). RNA polymerase III limits longevity downstream of TORC1. Nature, 552(7684):263-267 PubMed ID: 29186112

Marshall, L., Rideout, E. J., Grewal, S. S. (2012). Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila. EMBO J, 31(8):1916-1930 PubMed ID: 22367393

Michels, A. A., Robitaille, A. M., Buczynski-Ruchonnet, D., Hodroj, W., Reina, J. H., Hall, M. N., Hernandez, N. (2010). mTORC1 directly phosphorylates and regulates human MAF1. Mol Cell Biol, 30(15):3749-3757 PubMed ID: 20516213

Rideout, E. J., Marshall, L., Grewal, S. S. (2012). Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling. Proc Natl Acad Sci U S A, 109(4):1139-1144 PubMed ID: 22228302

Shekhar, A. C., Sun, Y. E., Khoo, S. K., Lin, Y. C., Malau, E. B., Chang, W. H., Chen, H. T. (2023). Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation. Nucleic Acids Res, 51(9):4223-4236 PubMed ID: 36484109

Sriskanthadevan-Pirahas, S., Deshpande, R., Lee, B., Grewal, S. S. (2018). Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila. PLoS Genet, 14(2):e1007202 PubMed ID: 29401457

Urena, E., Xu, B., Regan, J. C., Atilano, M. L., Minkley, L. J., Filer, D., Lu, Y. X., Bolukbasi, E., Khericha, M., Alic, N., Partridge, L. (2024). Trametinib ameliorates aging-associated gut pathology in Drosophila females by reducing Pol III activity in intestinal stem cells. Proc Natl Acad Sci U S A, 121(4):e2311313121 PubMed ID: 38241436

Weinmann, R., Roeder, R. G. (1974). Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. Proc Natl Acad Sci U S A, 71(5):1790-1794 PubMed ID: 4525293

Weinmann, R., Raskas, H. J., Roeder, R. G. (1974b). Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection. Proc Natl Acad Sci U S A, 71(9):3426-3439 PubMed ID: 4530313

Willemin, G., Mange, F., Praz, V., Lorrain, S., Cousin, P., Roger, C., Willis, I. M., Hernandez, N. (2023). Contrasting effects of whole-body and hepatocyte-specific deletion of the RNA polymerase III repressor Maf1 in the mouse. Front Mol Biosci, 10:1297800 PubMed ID: 38143800

date revised: 12 October, 2024

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