Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1
2009; Springer Nature; Volume: 28; Issue: 15 Linguagem: Inglês
10.1038/emboj.2009.179
ISSN1460-2075
AutoresYuehua Wei, Chi Kwan Tsang, Xiao-Feng Zheng,
Tópico(s)RNA modifications and cancer
ResumoArticle2 July 2009free access Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1 Yuehua Wei Yuehua Wei Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Chi Kwan Tsang Chi Kwan Tsang Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author X F Steven Zheng Corresponding Author X F Steven Zheng Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Yuehua Wei Yuehua Wei Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Chi Kwan Tsang Chi Kwan Tsang Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author X F Steven Zheng Corresponding Author X F Steven Zheng Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Author Information Yuehua Wei1,2, Chi Kwan Tsang2 and X F Steven Zheng 2 1Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA 2Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA *Corresponding author. Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, Staged Research Building, Room 142, 675 Hoes Lane, Piscataway, NJ 8854, USA. Tel.: +1 732 235 2894; Fax: +1 732 235 2875; E-mail: [email protected] The EMBO Journal (2009)28:2220-2230https://doi.org/10.1038/emboj.2009.179 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III. Introduction Cell growth is a process by which the cell increases its size and mass through the synthesis of proteins and other macromolecules. Ribosome biogenesis is crucial for growth by producing ribosomes, the machinery for protein synthesis. It accounts for the majority of nuclear transcription (up to 80%) in a eukaryotic cell (Warner, 1999; Moss and Stefanovsky, 2002). The cell tightly controls ribosome biogenesis in response to different growth and stress conditions. Deregulation of ribosome biogenesis is a prominent feature of many tumour cells: transformed cells overexpress the products of polymerase (Pol) I and III-transcribed genes; activation of Pol III-dependent transcription can promote oncogenic transformation (White, 2005; Johnson and Johnson, 2008). Ribosome biogenesis requires close coordination of all three RNA polymerases to produce individual components in an equal ratio (Warner, 1999; Moss and Stefanovsky, 2002). How the activity of all three RNA polymerases is controlled in a well-coordinated manner is a very interesting question. Recently, significant progress has been made that shows the cell has the intrinsic ability to balance the production of different ribosomal RNAs (rRNAs) (Laferte et al, 2006). However, the coordination of ribosome biogenesis is likely to be very complex and requires further understanding. TOR (target of rapamycin) is an evolutionally conserved PI3K-related kinase and a central regulator of cell growth (Wullschleger et al, 2006; Tsang et al, 2007b) originally found in budding yeast (Heitman et al, 1991). TOR proteins form two functional complexes. In yeast, TOR complex 1 (TORC1) consists of Kog1, Lst8, and either Tor1 or Tor2, whereas TOR complex 2 (TORC2) consists of Avo1-3, Lst8 and Tor2 (Loewith et al, 2002). The anticancer drug rapamycin specifically inhibits TORC1 after forming a complex with its intracellular receptor FKBP12 (FK506-binding protein 12 kDa), and the subsequent binding of FKBP12-rapamycin to FRB domain of TOR (Zheng et al, 1995). Mutations of Tor1 at Ser1972 block FKBP12-rapamycin binding and confer rapamycin resistance (Zheng et al, 1995). TORC1 regulates a broad spectrum of growth-related processes including ribosome biogenesis, protein translation, nutrient import and autophagy (Wullschleger et al, 2006; Tsang et al, 2007b). TORC2 carries out a rapamycin-insensitive essential function of regulating actin cytoskeleton organization (Loewith et al, 2002). TORC1 is a major regulator of transcription in yeast, including all ribosomal genes transcribed by three major RNA polymerases (Warner, 1999; Moss and Stefanovsky, 2002). Rapamycin and nutrient starvation cause rapid repression of ribosomal genes (Zaragoza et al, 1998; Powers and Walter, 1999). TOR is found in the cytoplasm as well as in the nucleus in mammals and yeast (Zhang et al, 2002; Drenan et al, 2004; Li et al, 2006), suggesting that TOR is likely to have nuclear functions such as gene regulation. Interestingly, TORC1 nuclear localization is essential for Pol I- but not Pol II-transcribed genes in yeast (Li et al, 2006). TORC1 in either cytoplasmic or nuclear form is sufficient to control Pol II gene transcription. TORC1 is further found to be associated with 35S promoter and 5S rDNA chromatins. This association is critically dependent on a helix-turn-helix (HTH) sequence, a classical DNA-binding motif. Interestingly, HTH deletion abolishes Tor1 association with rDNA chromatin but does not affect Tor1 localization in the nucleus, suggesting that this mutation specifically inhibits the ability of Tor1 to bind to rDNA chromatin. Together, these observations show that TORC1 association with rDNA chromatins is crucial for TORC1 to regulate 35S rDNA transcription. However, the significance of TORC1 association with rDNA chromatin in the regulation of Pol III-dependent genes, including 5S rDNA and tDNAs, remains to be established. Maf1 is a key regulator of Pol III-dependent transcription (Geiduschek and Kassavetis, 2006; Willis and Moir, 2007) that was initially identified in yeast (Murawski et al, 1994). It was later found to be a negative regulator of Pol III-dependent transcription (Boguta et al, 1997; Pluta et al, 2001). Mammalian Maf1 has also been recently reported to have similar functions (Johnson et al, 2007; Goodfellow et al, 2008), indicating that Maf1 proteins are evolutionarily conserved. Maf1 is a major effector mediating diverse growth and stress signals to control the transcription of Pol III-dependent genes, including 5S rRNA tRNA genes (Upadhya et al, 2002). It does so by preventing Pol III occupancy on its targeted genes, likely as a result of Maf1 interaction with Pol III (Desai et al, 2005). Maf1 is a phosphoprotein present in the cytoplasm under nutrient-rich conditions (Moir et al, 2006; Oficjalska-Pham et al, 2006; Roberts et al, 2006). In response to TORC1 inhibition by starvation or rapamycin, Maf1 becomes dephosphorylated and accumulates in the nucleus, resulting in association with and repression of 5S rDNA and tDNA (tRNA gene). Although these observations clearly show that TORC1 regulates Maf1 phosphorylation, the detailed mechanisms such as how TORC1 acts on Maf1 remain obscure. Moreover, how Maf1 activity is controlled remains unresolved. Protein kinase A (PKA) has been shown to phosphorylate Maf1 N-terminus in vitro and regulate Maf1 nuclear localization (Moir et al, 2006). However, in a strain lacking Maf1 nuclear exportin, Maf1 is constitutively nuclear but Pol III transcription remains normally regulated by TORC1 (Towpik et al, 2008), indicating that there is an unknown mechanism by which TORC1 controls Maf1 activity. In this study, we confirm that TORC1 is indeed associated with 5S rDNA and show that the ability of TORC1 to associate with rDNA chromatin is crucial for TORC1 to regulate Maf1 phosphorylation, and 5S rRNA and tRNA synthesis by Pol III. We further find that TORC1 is associated with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus relocalization. Together, these results show that TORC1 regulates Maf1 phosphorylation and subnuclear localization, and Pol III-dependent transcription in a chromatin-dependent mechanism. Results TORC1 association with 5S rDNA chromatin in a nutrient-dependent and rapamycin-sensitive manner In addition to 35S promoter chromatin, we showed earlier that Tor1 is associated with 5S rDNA chromatin in a rapamycin-sensitive manner (Li et al, 2006) (Figure 1A and C), suggesting a similar role of TORC1 in Pol III regulation. We, therefore, further characterized TORC1 association with 5S rDNA and the functional significance. We found that Tor1 association with 5S rDNA was significantly reduced by starvation (Figure 1B). Little or no chromatin immunoprecipitation (ChIP) signal was detected in a tor1Δ strain, indicating the specificity of the ChIP result. Rapamycin-sensitive association of Tor1 with 5S rDNA and 35S promoter (35S-P) was further confirmed by real-time PCR (Figure 1D). In contrast, the repetitive CUP1 promoter, and intergenic spacer 1 (IGS1) and 35S rDNA coding region did not show any significant Tor1 binding. Figure 1.TORC1 binds to 5S rDNA chromatin in a nutrient-dependent and rapamycin-sensitive manner. (A) Shown is the structure of a yeast rDNA repeat. N1-5 indicates the PCR primer sets used for ChIP assays. N4 covers 35S rDNA promoter region. (B) Tor1 associates with 5S rDNA and 35S promoter chromatin regions in a nutrient-dependent manner. Early log phase W303a wild-type (WT) and tor1Δ cells were starved for 30 min by nutrient depletion. ChIP assay was conducted using a Tor1-specific antibody. (C) Association of Tor1 with 5S rDNA and 35S promoter (35S-P) in a rapamycin-sensitive manner. Early log phase W303a WT and tor1Δ cells were treated without or with 100 nM rapamycin. ChIP assay was conducted with the PCR primer sets N2 and N4. (D) Real-time PCR quantification of Tor1 occupancy on various regions of rDNA sequences in the absence or the presence of rapamycin. ChIP samples were analysed by real-time PCR. Values are the average of three quantifications. Error bars represent the standard deviation. Fold enrichments were determined by comparing the ChIP signals of target regions with that of control (CUP1 promoter region). IGS1, 5S rDNA, 35S rDNA promoter (35S-P), 35S rDNA coding region (35S) were tested. (E) TORC1 but not TORC2 is associated with 5S rDNA and 35S promoter chromatin regions. Early log phase cells expressing Kog1-Myc9, Avo2-Myc9 or Avo3-Myc9 were treated without or with 100 nM rapamycin. ChIP assay was conducted by immunoprecipitation with a Myc-specific antibody (9E10) and the PCR primer set N2 and N4. (F) Real-time PCR quantification of Tor1 occupancy on various tDNA loci. ChIP samples were analysed by real-time PCR. Values are the average of three quantifications. Error bars represent the standard deviation. Fold enrichments were determined by comparing the ChIP signals of target regions with that of controls (ACT1 promoter region). A ribosome subunit gene (RPL9A) promoter was included as an unrelated control. (G) Nuclear localization controls TORC1 binding to 5S rDNA chromatin. Early log phase W303a WT and tor1Δ cells carrying a vector control or expressing Tor1-RR variants were starved for 30 min. The binding of WT Tor1, Tor1-RR, Tor1-RR/NLSmt and Tor1-RR/NESΔ to 5S rDNA was detected by ChIP assay with a Tor1-specific antibody and the PCR primer set N2. (H) HTH motif is required for TORC1 to bind to 5S rDNA. W303a tor1Δ cells carrying a vector control or expressing Tor1-RR or Tor1-RR/HTHΔ were cultured to early log phase and ChIP assay was conducted with a Tor1-specific antibody and the PCR primer set N2. Download figure Download PowerPoint We also investigated which TOR complex is associated with 5S rDNA. We found that Kog1-Myc9 was associated with 5S rDNA in a rapamycin-sensitive manner (Figure 1E) similar to what was observed with 35S rDNA promoter. In contrast, Avo2-Myc9 and Avo3-Myc9 were not detected in these chromatin regions. Thus, TORC1 but not TORC2 is associated with 5S rDNA and 35S promoter in a nutrient-dependent and rapamycin-sensitive manner. As Pol III is responsible for the synthesis of both tRNAs and 5S rRNA, we investigated the association of Tor1 with tRNA genes (tDNA). To our surprise, we were unable to detect significant Tor1 association with tDNA over the background (ACT1, RPL9A), as judged by both conventional and real-time PCR (Figure 1F and data not shown). Unlike the rRNA genes, TORC1 does not seem to be associated with tDNA chromatin. Association with 5S rDNA chromatin requires Tor1 nuclear localization and the HTH motif TORC1 is normally localized in both the nucleus and cytoplasm. On nutrient starvation, however, TORC1 is rapidly excluded from the nucleus (Li et al, 2006). Tor1 nuclear import and export are mediated by the nuclear localization sequence (NLS) and nuclear export sequence (NES), respectively. In addition, Tor1's ability to bind to 35S promoter is dependent on an HTH motif. Mutations in these motifs specifically block Tor1 nucleocytoplasmic transport or association with 35S rDNA promoter (Li et al, 2006). When such a mutation is combined with a rapamycin-resistant (RR) mutation, it provides a valuable tool to delineate the underlying physiological function of Tor1 localization or association with rDNA. For example, Tor1-RR/NLSmt is excluded from the nucleus, preventing it from association from 35S promoter (Li et al, 2006). In contrast, Tor1-RR/NESΔ, concentrated in the nucleus, is capable of binding to 35S rDNA promoter in a starvation-insensitive manner. In addition, HTHΔ disrupts the ability of Tor1-RR to associate with 35S rDNA promoter. Interestingly, both Tor1-RR/NLSmt and Tor1-RR/NLSΔ retain the ability to regulate Pol II-dependent transcription, revealing distinct mechanisms for Tor1 to control different class of genes (Li et al, 2006). To determine the requirement for TORC1 association with 5S rDNA chromatin, we studied Tor1-RR, Tor1-RR/NLSmt and Tor1-RR/NESΔ. We found that both endogenous wild-type Tor1 and Tor1-RR were associated with 5S rDNA chromatin in a nutrient-dependent manner (Figure 1G). Tor1-RR/NESΔ but not Tor1-RR/NLSmt was also found at 5S rDNA chromatin. Notably, the amount of Tor1-RR/NESΔ bound to 5S rDNA chromatin was considerably more than Tor1-RR, and this binding was resistant to nutrient starvation, indicating that Tor1 association with 5S rDNA chromatin is primarily regulated by Tor1 nuclear import. Tor1-RR/HTHΔ also failed to bind to 5S rDNA chromatin (Figure 1H). These observations show that TORC1 association with 5S rDNA chromatin is regulated by its nuclear localization and is dependent on the HTH motif. Strikingly, TORC1 shows the same requirement towards both 5S rDNA and 35S promoter chromatin, suggesting that TORC1 binding to both 5S and 35S rDNA chromatins is well coordinated. Regulation of Pol III-dependent transcription requires TORC1 nuclear localization and chromatin association TORC1 proteins in the nucleus and cytoplasm have distinct functions in the regulation of Pol I and Pol II genes: TORC1 nuclear localization and association with 35S promoter chromatin are required for the regulation of Pol I- but not Pol II-transcribed genes (Li et al, 2006). We wondered whether the control of Pol III-transcribed genes has similar requirements and addressed this question with different Tor1-RR variants with distinct localizations. Tor1-RR contains the Ser1972 → Ile mutation that prevents the binding of FKBP12-rapamycin to Tor1, conferring dominant rapamycin-resistance (Zheng et al, 1995). On adding rapamycin, endogenous TORC1 is knocked down, whereas TORC1 consisting of a Tor1-RR variant is not affected (Figure 2A), allowing specific analysis of these variants for their regulatory functions. In the absence of rapamycin, endogenous TORC1 (containing either Tor1 or Tor2) and the TORC1-containing Tor1-RR are capable of promoting ribosome biogenesis (Figure 2A a). In the presence of rapamycin, however, endogenous TORC1, but not the Tor1-RR-containing TORC1, is chemically knocked down by rapamycin (Figure 2A b). In the absence of rapamycin treatment, cells expressing Tor1-RR variants have normal ribosome biogenesis because of the presence of functional endogenous TORC1 (Figure 2A c) (Li et al, 2006). When treated with rapamycin, endogenous TORC1 is inhibited, whereas TORC1 containing a Tor1-RR variant (NESΔ, NLSmt or HTHΔ) is not affected (Figure 2A d), allowing us to specifically examine the effect of different mutations (NESΔ, NLSmt or HTHΔ) on ribosome biogenesis (Figure 2A d). It is worth noting that all three Tor1-RR variants still retain the ability to regulate Pol II-transcribed genes and Gln3 phoshorylation, indicating that they can form an otherwise functional TORC1 complex necessary for Gln3 regulation (Li et al, 2006). Figure 2.Transcription of Pol III-dependent genes requires TORC1 nuclear localization and its ability to associate with chromatin. (A) A strategy to study the regulation of ribosome biogenesis by TORC1. See main text for detailed description. (B) Synthesis of rRNAs and tRNAs in yeast cells expressing Tor1-RR variants. Early log phase W303a tor1Δ cells carrying vector control or expressing Tor1-RR variants were treated without or with rapamycin for 30 min, and then metabolically labelled with [5, 6-3H]-Uracil. Newly synthesized rRNAs and tRNAs were detected by autoradiography (upper panel). Total RNAs were stained by ethidium bromide (lower panel). (C) Transcription of pre-tRNALeu3 in yeast cells expressing Tor1-RR variants. Early log phase W303a tor1Δ cells carrying vector control or expressing Tor1-RR variants were treated without or with rapamycin for 30 min. The level of pre-tRNALeu3 was determined by northern blot with U4 small nuclear RNA as a loading control. The numbers represent the ratio of transcript levels after and before rapamycin treatment. Download figure Download PowerPoint To analyse the functions of different Tor1-RR variants, we metabolically labelled yeast cells with 3H-Uracil to monitor rRNA synthesis. In the absence of rapamycin, 5.8S rRNA (transcribed by Pol I) and 5S rRNA (transcribed by Pol III) were actively synthesized because of the presence of the endogenous TORC1 (Figure 2B). On rapamycin treatment, 5S and 5.8S rRNA synthesis was strongly inhibited in the control cells, or cells expressing Tor1-RR/NLSmt or Tor1-RR/HTHΔ. In contrast, 5S and 5.8S rRNA synthesis remained relatively normal in cells expressing Tor1-RR and Tor1-RR/NESΔ. The synthesis of tRNAs showed essentially the same behaviour as 5S and 5.8S rRNAs. To verify this result, we performed northern blot to detect labile precursor tRNA species (pre-tRNALeu3), which is another method to monitor tRNA synthesis (Upadhya et al, 2002; Oficjalska-Pham et al, 2006). Indeed, tRNA synthesis showed the similar response to the TOR1-RR variants (Figure 2C). These results indicate that nuclear localization and association with rDNA chromatin are required for Tor1 to control Pol III-dependent transcription. It is interesting to note that unlike 5S rRNA, tRNA synthesis is only moderately affected by rapamycin (Figure 2), which is consistent with earlier observations (Upadhya et al, 2002; Oficjalska-Pham et al, 2006). Maf1 phoshorylation and cytoplasmic localization depend on TORC1 nuclear localization and chromatin association Maf1 negatively regulates Pol III-dependent transcription in response to nutrient limitation or rapamycin treatment. Maf1 is a phosphoprotein whose phosphorylation is regulated by TORC1 (Moir et al, 2006; Oficjalska-Pham et al, 2006; Roberts et al, 2006). We investigated where Maf1 phosphorylation occurs using different Tor1-RR variants. In agreement with earlier studies (Moir et al, 2006; Oficjalska-Pham et al, 2006; Roberts et al, 2006), rapamycin inhibited Maf1 phosphorylation in the control cells of both S288C/FM391 and W303a strains (Figure 3A and B). In cells expressing Tor1-RR or Tor1-RR/NESΔ, however, Maf1 remained phosphorylated under the same condition. In contrast, rapamycin inhibited Maf1 phosphorylation in cell expressing Tor1-RR/NLSmt and Tor1-RR/HTHΔ, indicating that Maf1 phosphorylation critically depends on TORC1 nuclear localization and chromatin association. Figure 3.TORC1 regulates Maf1 phoshorylation and nucleocytoplasmic transport. (A) Maf1 phosphorylation requires TORC1 nuclear localization and chromatin association in S288C/FM391 cells. Exponentially growing S288C/FM391 cells carrying a vector control only or expressing Tor1-RR variants were treated without or with 100 nM rapamycin for 30 min. Phosphorylation of Maf1-Myc9 was determined by electrophoretic mobility (arrows) by western blot with a Myc-specific antibody. (B) Maf1 phosphorylation requires TORC1 nuclear localization and chromatin association in W303a cells. Exponentially growing W303a cells carrying a vector control only expressing Tor1-RR variants were treated without or with 100 nM rapamycin for 30 min. Phosphorylation of Maf1-Myc9 was determined by electrophoretic mobility (arrows) by western blot with a Myc-specific antibody. (C) Maf1 is predominantly cytoplasmic and rapamycin treatment causes Maf1 nuclear accumulation in S288C/FM391 cells. Exponentially growing S288C/FM391 cells carrying a vector control only or expressing Tor1-RR variants were treated without or with 100 nM rapamycin for 30 min. Maf1-Myc9 localization was determined by indirect immunofluorescence (IF) with a Myc-specific antibody. The nucleus was stained by DAPI. Download figure Download PowerPoint Maf1 is normally localized in the cytoplasm and rapamycin causes Maf1 to enter the nucleus, which has been proposed to control Maf1 activity (Moir et al, 2006; Oficjalska-Pham et al, 2006; Roberts et al, 2006). To further characterize the role of TORC1 regulation, we investigated Maf1 localization in cells expressing different Tor1-RR variants. In agreement with earlier findings, exponentially growing S288C/FM391 cells showed Maf1 distribution in the cytoplasm (Figure 3C). Interestingly, Maf1 was also clearly detectable in the nucleus. Rapamycin treatment caused Maf1 to enrich in the nucleus (Figure 3C). The same phenomenon was observed in cells expressing Tor1-RR/NLSmt and Tor1-RR/HTHΔ. In contrast, Maf1 remained throughout the entire cell in the presence of rapamycin in cells expressing Tor1-RR or Tor1-RR/NESΔ. Thus, regulation of Maf1 cytoplasmic retention requires TORC1 nuclear localization and rDNA chromatin association. It has been reported that artificially forced enrichment of Maf1 in the nucleus is insufficient to inhibit Pol III transcription of tRNA (Moir et al, 2006; Willis and Moir, 2007). In S288C/FM391 cells lacking the Maf1 nuclear exportin Msn5, Maf1 is enriched in the nucleus but Maf1 phosphorylation and Pol III-dependent transcription remain fully regulated by nutrient availability (Towpik et al, 2008). These observations suggest that there is a novel regulatory mechanism for Maf1 within the nucleus. Interestingly, we found that in W303a cells, Maf1 was constitutively nuclear (Supplementary Figure 1). However, Maf1 remained phosphorylated in a rapamycin-sensitive manner (Figure 3B). In addition, Pol III-dependent transcription in these cells was regulated in a rapamycin-sensitive manner (Figure 2B and C). Maf1 nuclear regulation occurs in both S288C/FM391 and W303, two most popular laboratory strains, indicating that the nuclear mechanism is a common regulation and is not strain specific. Thus, W303 strain provides an excellent model to investigate nuclear regulation of Maf1. One key difference between S288C/FM391 and W303 is that S288C/FM391 contains an allele of SSD1 called SSD1-v, whereas W303 strain carries an SSD1-d allele, based on their ability to suppress the lethality of sit4Δ mutation (Sutton et al, 1991). Interestingly, overexpression of SSD1 suppresses RNA Pol III mutations (Stettler et al, 1993), raising the possibility that Ssd1 is involved in Maf1 regulation. To test this hypothesis, we cloned SSD1-v from S288C/FM391 into a centromere plasmid and introduced it into W303a. Indeed, SSD1-v suppressed the rapamycin hypersensitive phenotype of the W303a tor1Δ strain (Supplementary Figure 2A), confirming an earlier observation (Reinke et al, 2004). In W303a cells carrying the SSD1 plasmid, there was a significant increase in Maf1 proteins in the cytoplasm (Supplementary Figure 2B), suggesting that Ssd1 is involved in the regulation of Maf1 cytoplasmic localization. TORC1 controls Maf1 nucleolar localization and association with Pol III-dependent genes The nucleolus is a subnuclear compartment in which 5S rDNA is transcribed by Pol III. We found that Pol III-specific subunit Rpc82 was distributed throughout the entire nucleus, which did not change significantly in response to rapamycin treatment (Figure 4A; see Supplementary Figure 3A for colour images). Thus, a simple mechanism for TORC1 to regulate Pol III activity and Maf1 within the nucleus is to control Maf1's accessibility to the nucleolus. Indeed, we therefore investigated Maf1 localization in W303a cells and found that Maf1 in exponential W303a cells was localized in the nucleus but excluded from the nucleolus (Figure 4B–D; see Supplementary Figure 3B and C for colour images). After rapamycin treatment, however, Maf1 rapidly entered the nucleolus as indicated by co-staining with Nop1, a nucleolar marker. Thus, TORC1 normally keeps Maf1 outside the nucleolus. We further studied Maf1 association with Pol III-transcribed genes by ChIP assay. In exponentially growing cells, Maf1 was poorly associated with 5S rDNA and tDNA (Figure 5A and B, 0 min). On rapamycin treatment, however, Maf1 occupancy at these chromatin regions increased significantly. Interestingly, Maf1 association with 5S rDNA and tDNA chromatins peaked at around 20 min of rapamycin treatment and then tapered (Figure 5), indicating that Maf1 only transiently associates with Pol III-transcribed genes. The peak Maf1 occupancy at Pol III-transcribed genes occurred earlier in W303a than S288C/FM391 cells. As Maf1 is already localized in the nucleus in W303a cells, a logic explanation for this phenomenon is that it takes less time for Maf1 to travel to the nucleolus in this genetic background. The presence of Tor1-RR or Tor1-RR/NESΔ, but not Tor1-RR/HTHΔ or Tor1-RR/NLSmt, prevented the rapamycin-induced increase of Maf1 association with Pol III-transcribed genes (Figures 4E and 5C), suggesting that TORC1 regulates Maf1 occupancy on Pol III-dependent genes within the nucleus and at rDNA chromatin. Figure 4.Maf1 is normally excluded from the nucleolus and inhibition of TORC1 promotes Maf1 nucleolar localization and 5S rDNA association. (A) Rpc82 is localized in both nucleus and nucleolus, which is not affected by rapamycin treatment. Exponentially growing W303a cells expressing Rpc82-Myc9 were treated without or with 100 nM rapamycin for 30 min. Rpc82-Myc9 localization was determined by IF with a Myc-specific antibody. The nucleolus was stained with a Nop1 antibody and the nucleus was stained by DAPI. (B) Maf1 is normally absent from the nucleolus and rapamycin causes Maf1 nucleolar localization. Exponentially growing W303a cells expressing Maf1-Myc9 were treated without or with 100 nM rapamycin for 30 min. Maf1-Myc9 localization was determined by IF with a Myc-specific antibody. The nucleolus was stained with a Nop1 antibody and the nucleus was stained by DAPI. (C) Shown are enlarged images of representative cells from Figure 4B. The dotted line indicates the nucleoplasm. (D) Quantification of cells with different Maf1 localization (white bar, cells with Maf1 in the nucleolus; black bar, cells with Maf1 excluded from the nucleolus) (N⩾200). (E) Inhibition of Maf1 association with 5S rDNA chromatin is dependent on TORC1 nuclear localization and rDNA chromatin binding. W303a cells carrying a vector control or expressing Tor1-RR variants were treated without and with 100 nM rapamycin and ChIP assay was performed with a Myc-specific antibody. Download figure Download PowerPoint Figure 5.Inhibition of TORC1 causes transient Maf1 association
Referência(s)