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168 Biochemical Society Transactions (2012) Volume 40, part 1 Roles of the mammalian target of rapamycin, mTOR, in controlling ribosome biogenesis and protein synthesis Valentina Iadevaia*, Yilin Huo*, Ze Zhang*, Leonard J. Foster† and Christopher G. Proud*1 *School of Biological Sciences, Life Sciences Building, Highfield Campus, University of Southampton, Southampton SO17 1BJ, U.K., and †Department of Biochemistry and Molecular Biology and Centre for High-Throughput Biology, 2125 East Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4 Abstract mTORC1 (mammalian target of rapamycin complex 1) is controlled by diverse signals (e.g. hormones, growth factors, nutrients and cellular energy status) and regulates a range of processes including anabolic metabolism, cell growth and cell division. We have studied the impact of inhibiting mTOR on protein synthesis in human cells. Partial inhibition of mTORC1 by rapamycin has only a limited impact on protein synthesis, but inhibiting mTOR kinase activity causes much greater inhibition of protein synthesis. Using a pulsed stable-isotope-labelling technique, we show that the rapamycin and mTOR (mammalian target of rapamycin) kinase inhibitors have differential effects on the synthesis of specific proteins. In particular, the synthesis of proteins encoded by mRNAs that have a 5 -terminal pyrimidine tract is strongly inhibited by mTOR kinase inhibitors. Many of these mRNAs encode ribosomal proteins. mTORC1 also promotes the synthesis of rRNA, although the mechanisms involved remain to be clarified. We found that mTORC1 also regulates the processing of the precursors of rRNA. mTORC1 thus co-ordinates several steps in ribosome biogenesis. Introduction mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase. mTOR interacts with other proteins to form two distinct types of complex termed mTORC (mTOR complex) 1 and 2. In the short term, rapamycin, a macrolide antibiotic, inhibits some, but not all, of the functions of mTORC1. In some cell types, over longer treatment times, rapamycin can also interfere with mTORC2 [1]. mTORC1 and mTORC2 have distinct substrates, and this specificity is likely to be conferred by the partner proteins raptor (regulatory associated protein of mTOR) (mTORC1) and rictor (rapamycin-insensitive companion of mTOR) (mTORC2). For example, raptor interacts with substrates for mTORC1 through their TOS (target of rapamycin signalling) motifs. These substrates include S6K (ribosomal S6 kinase) 1 and 2, and the 4E-BPs [eIF (eukaryotic initiation factor) 4E-binding proteins]. Substrates for mTORC2 include PKB (protein kinase B) (also called Akt) and a related kinase, SGK (serum- and glucocorticoid-inducible kinase). S6Ks are implicated in the control of cell and organismal growth [2–4]. Control of protein synthesis makes an Key words: mammalian target of rapamycin (mTOR), mRNA, protein synthesis, rapamycin, ribosome biogenesis, translation factor. Abbreviations used: eIF, eukaryotic initiation factor; 4E-BP, eIF4E-binding protein; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; mTOR-KI, mTOR kinase inhibitor; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; Pol, RNA polymerase; raptor, regulatory associated protein of mTOR; rictor, rapamycin-insensitive companion of mTOR; S6K, ribosomal S6 kinase; TFIIIC, transcription factor IIIC; TOP, terminal tract of pyrimidines; TOS, target of rapamycin signalling. 1 To whom correspondence should be addressed (email [email protected]). C The C 2012 Biochemical Society Authors Journal compilation important contribution to cell growth and division. This is for several reasons: (i) the bulk of the dry mass of cells is protein, so increased cell growth or cell division generally requires faster overall protein synthesis, and (ii) cell division, for example, requires specific proteins involved in checkpoints during the cell cycle, and their production is key to cell-cycle progression. Many mitogenic, anabolic or hypertrophic stimuli activate protein synthesis. Initially (within minutes or a few hours), this involves the activation of existing components of the translational machinery, such as translation factors, and mainly the enhanced translation of existing mRNAs (i.e. improved ‘translational efficiency’). Over longer times (hours to days), additional mRNAs will be transcribed and the cell will generate more ribosomes, through activated ribosome biogenesis, as well as translation factors. Thus, over these longer times, there is an increase in the translational capacity of the cell. The S6Ks phosphorylate several substrates which are linked to protein synthesis (mRNA translation) including ribosomal protein S6, a component of the 40S ribosomal subunit, eEF2K (eukaryotic elongation factor 2 kinase) [5], eIF4B (which promotes the activity of an RNA helicase, eIF4A) [6] and SKAR (S6K1 Aly/REF-like target) [7] (which is related to proteins involved in pre-mRNA splicing and mRNA transport). However, it remains unclear what contribution the phosphorylation of these S6K substrates makes to the control of mRNA translation. The 4E-BPs interact with eIF4E, the protein that binds the 5 -cap structure of cytoplasmic mRNAs, and prevent Biochem. Soc. Trans. (2012) 40, 168–172; doi:10.1042/BST20110682 Signalling 2011: a Biochemical Society Centenary Celebration Figure 1 Control of translational efficiency and capacity by mTORC1 The Figure summarizes the main known mechanisms by which signalling downstream of mTORC1 regulates translational capacity and efficiency (e.g. ribosome levels). This occurs through regulation of mRNA translation in the cytoplasm, Pol III in the nucleoplasm and Pol I in the nucleoli (grey region). Black arrows indicate established links and grey arrows connections that are not fully understood. Question marks denote likely connections or unidentified linking components. See the text for further details. phosphorylation of S6 itself, but subsequent studies have disproved this [11,12]. Secondly, mTORC1 promotes the transcription of rRNAs, which is catalysed by Pol III (5S rRNA) or Pol I (the other three rRNAs) (reviewed in [13]). Although connections between mTORC1 and the transcriptional machinery that makes rRNA have been described (e.g. [14–16]), it is still not fully understood how mTORC1 controls rRNA synthesis (Figure 1). It is interesting to note that recent work has shown that mTORC2 associates with ribosomes [17,18], and appears to be controlled by this interaction, suggesting that ribosome number, for example, can control the activation of PKB signalling. Recently, a number of compounds that inhibit the kinase activity of mTOR have been developed, including PP242 [19] and AZD8055 [20]. These compounds, called mTOR-KIs (mTOR kinase inhibitors) inhibit mTORC1 and mTORC2. Their effects are thus expected to be distinct from those of rapamycin, which only interferes with some functions of mTORC1 [19,21]. We have examined the effects of mTORKIs and rapamycin on protein synthesis and the production of rRNA. mTOR-KIs inhibit overall protein synthesis more strongly than rapamycin it from engaging with the scaffold protein eIF4G to form initiation factor complexes. mTORC1 phosphorylates 4EBP1 at several sites, resulting in its release from eIF4E, allowing it to bind eIF4G. eIF4G also binds eIF4A, and formation of eIF4E–eIF4G–eIF4A complexes is thought to be especially important for efficient translation of mRNAs that contain secondary structure within their 5 -UTRs (untranslated regions) (Figure 1). Recent findings show that 4E-BPs link mTORC1 to the control of cell proliferation, whereas S6Ks control cell growth [2]. mTORC1 also regulates ribosome biogenesis; it does so in at least two main ways. First, it promotes the translation of the mRNAs encoding all of the cytoplasmic ribosomal proteins (and some other components of the translational machinery, such as elongation factors [8]). The translation of these mRNAs is regulated through a 5 -TOP (terminal tract of pyrimidines), which suppresses their translation under basal (e.g. serum-starved) conditions; their translation is increased, e.g., by serum (Figure 1). This effect is inhibited by rapamycin and more strongly by inhibitors of PI3K (phosphoinositide 3-kinase) such as LY294002 [9]. However, although the ability of rapamycin to impair 5 -TOP mRNA translation has been known for more than 15 years [10], it is not clear how mTORC1 controls their translation. At one time, this was thought to be mediated through the S6Ks and perhaps We studied the effects of rapamycin and mTOR-KIs in HeLa cells, using the standard approach of metabolic labelling of newly synthesized proteins with [35 S]methionine. In serumfed cells, treatment with rapamycin for 2 or 6 h had only a small effect on the rate of protein synthesis, inhibiting it by 10% or less. In contrast, mTOR-KIs exerted a much stronger inhibition, typically causing a 30–40% reduction in the rate of incorporation. This difference could reflect either a role for mTORC2 in the short-term control of translation or effects of mTORKIs on components downstream of mTORC1 which are not affected by rapamycin. The effects of rapamycin and PP242 were similar in cells lacking Sin1, and thus devoid of mTORC2, and wild-type cells [19]. This implies that the effects of mTOR-KIs on protein synthesis are mediated not through mTORC2, but through mTORC1. We therefore studied the effects of rapamycin and mTORKIs on proteins known to be regulated through mTORC1. Rapamycin completely inhibited S6 phosphorylation, implying (i) that phosphorylation of S6 (and of other substrates for the S6Ks) plays only a minor role, at most, in maintaining protein synthesis rates, and (ii) that the phosphorylation of these proteins cannot explain the differing effects of rapamycin and mTOR-KIs on translation rates. In contrast, rapamycin and mTOR-KIs had quite differing effects on the association of 4E-BP1 and eIF4G with eIF4E. Whereas the latter caused the dephosphorylation of 4E-BP1, resulting in its increased binding to eIF4E and loss of eIF4E– eIF4G complexes, rapamycin did not. Thus this branch of mTORC1 signalling is ‘resistant’ to rapamycin in HeLa cells. C The C 2012 Biochemical Society Authors Journal compilation 169 170 Biochemical Society Transactions (2012) Volume 40, part 1 In principle, the ability of mTOR-KIs, but not rapamycin, to block eIF4E–eIF4G binding could account for their differing effects on overall protein synthesis. Further work is needed to understand how mTOR-KIs inhibit ongoing protein synthesis and also the mechanism by which rapamycin exerts its modest inhibitory effect. Effects of mTOR-KIs and rapamycin on the synthesis of specific proteins To explore the effects of rapamycin and mTOR-KIs on the synthesis of specific proteins, we made use of a newly developed heavy-isotope-labelling procedure which we call pSILAC (pulsed stable-isotope labelling with amino acids in cell culture). At the start of the study period, cells are transferred to medium containing heavy-isotope (13 C or 15 N)-tagged versions of arginine and lysine. In our experiments, where appropriate, mTOR inhibitors were added 30 min before this. Newly synthesized proteins will contain ‘tagged’ versions of lysine and arginine. After cell lysis and tryptic digestion of proteins, peptides are analysed by MS, where the ‘light’ and ‘heavy’ versions of each peptide can be resolved, identified and quantified. The proportion of any given peptide present as the heavier form indicates the rate of accumulation of the parent protein from which it is derived. Analysis of the data reveals the impact of signalling inhibitors on the synthesis of specific polypeptides. Our results show that rapamycin and mTOR-KIs have different effects on the synthesis of many proteins. In general, mTOR-KIs inhibit their synthesis more strongly than does rapamycin, in line with the effects of these compounds on overall protein synthesis rates. For proteins known to be encoded by 5 -TOP mRNAs, such as ribosomal proteins, rapamycin inhibited synthesis quite strongly (typically by 20–50%), which is roughly in line with results from earlier studies (which mostly focused on the association of such mRNAs with polyribosomes rather than actual rates of synthesis of the corresponding proteins). In contrast, mTOR-KIs exerted much stronger inhibition, with inhibition of more than 50% being seen for most of these proteins. Importantly, this suggests that mTOR, probably mTORC1, but perhaps via another type of mTORC [22], controls the synthesis of these proteins by rapamycin-sensitive and -insensitive mechanisms. A key issue is to identify how mTORC1 modulates the translation of 5 -TOP mRNAs. The rapamycin-insensitive input to mTOR regulation of 5 -TOP mRNAs may contribute to the inhibitory effects of the PI3K inhibitors mentioned above, since LY294002 also inhibits mTOR activity [23]. Rapamycin only rather weakly inhibited the synthesis of most other proteins, with the mTOR-KIs slowing their production by between 20 and 50%. Thus mTOR inhibition has less impact on the synthesis of most proteins that are not encoded by non-5 -TOP mRNAs. Interestingly, within the set of proteins initially categorized as encoded C The C 2012 Biochemical Society Authors Journal compilation by non-5 -TOP mRNAs are a number whose synthesis is strongly affected by mTOR-KIs or rapamycin and which appear (as judged from the database of transcriptional start sites at http://dbtss.hgc.jp/) to contain 5 -terminal sequences resembling a TOP. These may be previously unrecognized 5 -TOP mRNAs. An earlier study pointed to the existence of many human mRNAs that contain a 5 -TOP, although, for most of them, this feature was not ‘conserved’ in mice [24]. Nevertheless, taken together, these data point to the possibility that signalling through mTORC1 may, via 5 TOP-mediated translational control, promote the synthesis of a larger number and diversity of proteins than has hitherto been recognized. This mechanism may therefore mediate the regulation by mTORC1 of other processes in addition to ribosome biogenesis and translational capacity. Control of ribosome biogenesis by mTOR As noted above, mTORC1 is known to control the transcription of rRNA as well as the synthesis of ribosomal proteins [13]. Rapamycin impairs the formation of transcriptioninitiation complexes involved in transcribing the 45S prerRNA genes [15]. The major rRNAs (5.8S, 18S and 28S) are derived from a single primary transcript, the 45S pre-rRNA, which is made by Pol (RNA polymerase) I. Rapamycin causes the inactivation of TIF-1A, a component of the Pol I transcriptional machinery, via changes in its phosphorylation. Rapamycin causes the dephosphorylation of one site in TIF1A and increased phosphorylation of another one. It also leads to accumulation of TIF-1A in the cytoplasm. It is important to note that mTORC1 has been shown to associate with the promoters for genes transcribed by Pol I and Pol III [25,26]. mTORC1 (and its yeast counterpart) are thought to phosphorylate components involved in Pol III function directly, but it is not clear how mTORC1 controls Pol I. Earlier work indicated that S6K1 played a role in promoting the activity of Pol I [16]. The mechanism by which rapamycin impairs rRNA transcription appears to involve dephosphorylation of the C-terminal tail of UBF (upstream binding factor), which binds directly to rDNA and interacts with other components of the RNA Pol I machinery, in particular SL-1 [16]. In the case of Pol III, which makes 5S rRNA, regulation by TORC1 in yeast involves the phosphorylation of Maf1, a repressor of Pol III [27]. Phosphorylated Maf1 is excluded from the nucleus, thus alleviating the inhibition of Pol I transcription. TORC1 appears to promote the phosphorylation of Maf1 both directly (i.e. it can phosphorylate Maf1) [28] and through the protein kinase Sch9, a homologue of the mammalian S6Ks [29–31]. The latter apparently mediates the nuclear exclusion of Maf1, whereas TORC1 regulates the association of Maf1 with chromatin [29]. Mammalian Maf1 undergoes dephosphorylation in response to inhibition of mTORC1 [26,32] and has been reported to be a direct substrate for mTORC1 which can phosphorylate it at several sites [32]. Knockdown Signalling 2011: a Biochemical Society Centenary Celebration or knockout of Maf1 rendered Pol III-mediated tRNA transcription insensitive to inhibition of mTORC1 [26,32], confirming its importance for the control of Pol II by mTOR. In the case of mammalian Maf1, which does not contain an obvious nuclear localization signal, mTORC1 mainly acts by ensuring the exclusion of Maf1 from chromatin rather than from the nucleus, which contrasts with the situation in yeast. Another difference between yeast and mammalian Maf1 is that the Sch9 phosphorylation sites in the yeast protein are absent from the shorter mammalian orthologue. However, a rapamycin-resistant mutant of S6K1 can partially reverse the dephosphorylation of Maf1 caused by mTOR inhibition [26]. Kantidakis et al. [14] have confirmed that mTOR associates with Pol III genes and have shown that it binds to TFIIIC (transcription factor IIIC), which binds to Pol III promoters. This mechanism for localization is likely to be mediated through a TOS motif in the TFIIIC component TFIIIC63, thereby allowing mTORC1 to phosphorylate Maf1 on chromatin. The 45S pre-rRNA must be processed, through multiple steps, to yield the mature 5.8S, 18S and 28S rRNAs. Our own recent results suggest that mTORC1 also regulates this process. Treatment of cells with rapamycin causes the decay of newly made rRNA and the accumulation of intermediates in the rRNA-processing pathway, indicating that it blocks specific steps in that process. Concluding comments Although important advances continue to be made in understanding how mTOR controls cell function, there remain important gaps in our understanding. The present article has highlighted the fact that we do not understand how it regulates steps involved in ribosome biogenesis, such as the translation of 5 -TOP mRNAs, the activity of Pol I or the processing of rRNA. It is also still unclear how mTORKIs impair general protein synthesis in HeLa cells, given that this seems not to be mediated through inactivation of eIF4E by hypophosphorylated 4E-BPs. Funding This work was supported by grants to C.G.P. from AstraZeneca, the European Union and the British Heart Foundation. References 1 Sarbassov, D.D., Ali, S.M., Sengupta, S., Sheen, J.H., Hsu, P.P., Bagley, A.F., Markhard, A.L. and Sabatini, D.M. (2006) Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol. Cell 22, 159–168 2 Dowling, R.J., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B.D., Petroulakis, E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y. et al. (2010) mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328, 1172–1176 3 Montagne, J., Stewart, M.J., Stocker, H., Hafen, E., Kozma, S.C. and Thomas, G. (1999) Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129 4 Shima, H., Pende, M., Chen, Y., Fumagalli, S., Thomas, G. and Kozma, S.C. (1998) Disruption of the p70S6k /p85S6k gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17, 6649–6659 5 Wang, X., Li, W., Williams, M., Terada, N., Alessi, D.R. and Proud, C.G. (2001) Regulation of elongation factor 2 kinase by p90RSK1 and p70 S6 kinase. EMBO J. 20, 4370–4379 6 Shahbazian, D., Roux, P.P., Mieulet, V., Cohen, M.S., Raught, B., Taunton, J., Hershey, J.W., Blenis, J., Pende, M. and Sonenberg, N. (2006) The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J. 25, 2781–2791 7 Jastrzebski, K., Hannan, K.M., Tchoubrieva, E.B., Hannan, R.D. and Pearson, R.B. (2007) Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors 25, 209–226 8 Iadevaia, V., Caldarola, S., Tino, E., Amaldi, F. and Loreni, F. (2008) All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5 -terminal oligopyrimidine (TOP) mRNAs. RNA 14, 1730–1736 9 Stolovich, M., Tang, H., Hornstein, E., Levy, G., Cohen, R., Bae, S.S., Birnbaum, M.J. and Meyuhas, O. (2002) Transduction of growth or mitogenic signals into translational activation of TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but requires neither S6K1 nor rpS6 phosphorylation. Mol. Cell. Biol. 22, 8101–8113 10 Jefferies, H.B.J., Reinhard, G., Kozma, S.C. and Thomas, G. (1994) Rapamycin selectively represses translation of the ‘polypyrimidine tract’ mRNA family. Proc. Natl. Acad. Sci. U.S.A. 91, 4441–4445 11 Pende, M., Um, S.H., Mieulet, V., Sticker, M., Goss, V.L., Mestan, J., Mueller, M., Fumagalli, S., Kozma, S.C. and Thomas, G. (2004) S6K1 − / − /S6K2 − / − mice exhibit perinatal lethality and rapamycin-sensitive 5 -terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol. Cell. Biol. 24, 3112–3124 12 Ruvinsky, I., Sharon, N., Lerer, T., Cohen, H., Stolovich-Rain, M., Nir, T., Dor, Y., Zisman, P. and Meyuhas, O. (2005) Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 19, 2199–2211 13 Mayer, C. and Grummt, I. (2006) Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases. Oncogene 25, 6384–6391 14 Kantidakis, T., Ramsbottom, B.A., Birch, J.L., Dowding, S.N. and White, R.J. (2010) mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their repressor Maf1. Proc. Natl. Acad. Sci. U.S.A. 107, 11823–11828 15 Mayer, C., Zhao, J., Yuan, X. and Grummt, I. (2004) mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev. 18, 423–434 16 Hannan, K.M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A., Rothblum, L., Moss, T., Poortinga, G., McArthur, G.A., Pearson, R.B. and Hannan, R.D. (2003) mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF. Mol. Cell. Biol. 23, 8862–8877 17 Oh, W.J., Wu, C.C., Kim, S.J., Facchinetti, V., Julien, L.A., Finlan, M., Roux, P.P., Su, B. and Jacinto, E. (2010) mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J. 29, 3939–3951 18 Zinzalla, V., Stracka, D., Oppliger, W. and Hall, M.N. (2011) Activation of mTORC2 by association with the ribosome. Cell 144, 757–768 19 Feldman, M.E., Apsel, B., Uotila, A., Loewith, R., Knight, Z.A., Ruggero, D. and Shokat, K.M. (2009) Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 7, e38 20 Chresta, C.M., Davies, B.R., Hickson, I., Harding, T., Cosulich, S., Critchlow, S.E., Vincent, J.P., Ellston, R., Jones, D., Sini, P. et al. (2010) AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 70, 288–298 21 Wang, X., Beugnet, A., Murakami, M., Yamanaka, S. and Proud, C.G. (2005) Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins Mol. Cell. Biol. 25, 2558–2572 22 Patursky-Polischuk, I., Stolovich-Rain, M., Hausner-Hanochi, M., Kasir, J., Cybulski, N., Avruch, J., Ruegg, M.A., Hall, M.N. and Meyuhas, O. (2009) The TSC–mTOR pathway mediates translational activation of TOP mRNAs by insulin largely in a raptor- or rictor-independent manner. Mol. Cell. Biol. 29, 640–649 C The C 2012 Biochemical Society Authors Journal compilation 171 172 Biochemical Society Transactions (2012) Volume 40, part 1 23 Brunn, G.J., Williams, J., Sabers, C., Weiderrecht, G., Lawrence, J.C. and Abraham, R.T. (1996) Direct inhibition of the signalling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 15, 5256–5267 24 Yamashita, R., Suzuki, Y., Takeuchi, N., Wakaguri, H., Ueda, T., Sugano, S. and Nakai, K. (2008) Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics. Nucleic Acids Res. 36, 3707–3715 25 Tsang, C.K., Liu, H. and Zheng, X.F. (2010) mTOR binds to the promoters of RNA polymerase I- and III-transcribed genes. Cell Cycle 9, 953–957 26 Shor, B., Wu, J., Shakey, Q., Toral-Barza, L., Shi, C., Follettie, M. and Yu, K. (2010) Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells. J. Biol. Chem. 285, 15380–15392 27 Wei, Y. and Zheng, X.F. (2009) TORC1 association with rDNA chromatin as a mechanism to co-regulate Pol I and Pol III. Cell Cycle 8, 3802–3803 28 Wei, Y., Tsang, C.K. and Zheng, X.F. (2009) Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J. 28, 2220–2230 C The C 2012 Biochemical Society Authors Journal compilation 29 Wei, Y. and Zheng, X.F. (2009) Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8, 4085–4090 30 Huber, A., Bodenmiller, B., Uotila, A., Stahl, M., Wanka, S., Gerrits, B., Aebersold, R. and Loewith, R. (2009) Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev. 23, 1929–1943 31 Lee, J., Moir, R.D. and Willis, I.M. (2009) Regulation of RNA polymerase III transcription involves SCH9-dependent and SCH9-independent branches of the target of rapamycin (TOR) pathway. J. Biol. Chem. 284, 12604–12608 32 Michels, A.A., Robitaille, A.M., Buczynski-Ruchonnet, D., Hodroj, W., Reina, J.H., Hall, M.N. and Hernandez, N. (2010) mTORC1 directly phosphorylates and regulates human MAF1. Mol. Cell Biol. 30, 3749–3757 Received 4 August 2011 doi:10.1042/BST20110682