PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression

Article

PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression Graphical Abstract

Authors Ying Liu, Jaakko Mattila, €, ..., Markku Varjosalo, Sami Ventela Jukka Westermarck, Ville Hietakangas

Correspondence [email protected]

In Brief Ribosome biogenesis, which regulates animal growth, is controlled by nutrientresponsive mTOR signaling. Liu et al. uncover a role for chromatin-associated protein PWP1 as a downstream effector of mTOR signaling in this context in Drosophila. PWP1 associates with RNA polymerase I and regulates the expression of ribosomal RNAs in the nucleolus.

Highlights d

PWP1 is a nutrient-responsive regulator of animal growth

d

PWP1 associates with RNA polymerase I and regulates rRNA expression

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mTORC1 regulates PWP1 expression and phosphorylation

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High PWP1 expression in human tumors is associated with poor prognosis

Liu et al., 2017, Developmental Cell 43, 240–252 October 23, 2017 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.devcel.2017.09.022

Developmental Cell

Article PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression €,3 Leena Yadav,2 Wei Zhang,1,2,8 Nicole Lamichane,1,2 Jari Sundstro¨m,4,5 Ying Liu,1,2 Jaakko Mattila,1,2,7 Sami Ventela Otto Kauko,3,4 Reidar Gre´nman,6 Markku Varjosalo,2 Jukka Westermarck,3,4 and Ville Hietakangas1,2,9,* 1Department

of Biosciences, University of Helsinki, 00790 Helsinki, Finland of Biotechnology, University of Helsinki, 00790 Helsinki, Finland 3Turku Centre for Biotechnology, University of Turku and A ˚ bo Akademi University, 20520 Turku, Finland 4Department of Pathology, University of Turku, 20520 Turku, Finland 5Department of Pathology, Turku University Hospital, 20521 Turku, Finland 6Department of Otorhinolaryngology - Head and Neck Surgery and Department of Medical Biochemistry and Genetics, University of Turku and Turku University Hospital, 20521 Turku, Finland 7Present address: DKFZ, 69120 Heidelberg, Germany 8Present address: College of Animal Science and Technology, Nanjing Agriculture University, 210014 Nanjing, China 9Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2017.09.022 2Institute

SUMMARY

Ribosome biogenesis regulates animal growth and is controlled by nutrient-responsive mTOR signaling. How ribosome biogenesis is regulated during the developmental growth of animals and how nutrientresponsive signaling adjusts ribosome biogenesis in this setting have remained insufficiently understood. We uncover PWP1 as a chromatin-associated regulator of developmental growth with a conserved role in RNA polymerase I (Pol I)-mediated rRNA transcription. We further observed that PWP1 epigenetically maintains the rDNA loci in a transcriptioncompetent state. PWP1 responds to nutrition in Drosophila larvae via mTOR signaling through gene expression and phosphorylation, which controls the nucleolar localization of dPWP1. Our data further imply that dPWP1 acts synergistically with mTOR signaling to regulate the nucleolar localization of TFIIH, a known elongation factor of Pol I. Ribosome biogenesis is often deregulated in cancer, and we demonstrate that high PWP1 levels in human head and neck squamous cell carcinoma tumors are associated with poor prognosis.

INTRODUCTION The ability to coordinate growth rate with nutrient status is a fundamental feature of all organisms, yet our understanding of the underlying mechanisms remains incomplete. In multicellular animals, nutrition has a striking impact on body size. For example, severely nutrient-deprived Drosophila larvae display a prolonged larval period and emerge as adults with significantly smaller size than their well-fed siblings (Hietakangas and Cohen,

2009). By compromising in developmental timing and body size, the animal retains sufficient resources for the physiological functions that are essential for survival (Hietakangas and Cohen, 2009). Nutrient availability is intimately coupled with protein biosynthesis, which is a rate-limiting process in tissue growth. The cellular capacity for protein production is adjusted at €inen and multiple levels, including ribosome biogenesis (Lempia Shore, 2009). Ribosome biogenesis requires the activities of all three RNA polymerases (Mayer and Grummt, 2006). The rRNA species are transcribed by RNA polymerases (Pol) I and III (Lem€inen and Shore, 2009); Pol I acts in the nucleolus to transcribe pia the 47S pre-rRNA, which is processed into the 5.8S, 18S, and 28S rRNAs (Kos and Tollervey, 2010; Schneider et al., 2006; Tschochner and Hurt, 2003), while nucleoplasmic Pol III transcribes the 5S rRNA (Moir and Willis, 2013). Although ribosome biogenesis involves coordination of several activities, Pol I-mediated transcription is a key determinant of the rate of ribosome biogenesis (Laferte´ et al., 2006). Thereby, the nucleolus is an important signaling hub, converging pathways involved in growth control (Ruggero, 2012; Tsai and Pederson, 2014). Nucleolar rRNA expression is typically deregulated in cancer, and therefore new strategies to therapeutically target Pol I function are being developed (Bywater et al., 2013; Drygin et al., 2010; Peltonen et al., 2014). A key mediator of nutrient-dependent control of ribosome biogenesis is the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which integrates signals from amino acids and energy sensing with those of the insulin/insulin-like growth factors (Gentilella et al., 2015; Powers and Walter, 1999). mTORC1 activity is essential for Pol I- and Pol III-dependent rRNA transcription (Hannan et al., 2003; Powers and Walter, 1999; Zaragoza et al., 1998). In cultured mammalian cells, mTORC1 signaling has been shown to regulate the phosphorylation of transcription initiation factor 1A (TIF-1A) and upstream binding factor (UBF), which are essential activators of Pol I-dependent transcription (Hannan et al., 2003; Mayer et al., 2004). Genetic experiments in Drosophila have shown that TIF-1A is needed

240 Developmental Cell 43, 240–252, October 23, 2017 ª 2017 Elsevier Inc.

for TORC1-driven growth in vivo, while no clear functional ortholog for UBF is present in Drosophila (Grewal et al., 2007; Marshall et al., 2012). Furthermore, in Drosophila, mTOR signaling promotes the expression of transcription factor Myc (Teleman et al., 2008). Drosophila Myc facilitates nucleolar rRNA expression by promoting Pol II-mediated expression of TIF-1A and other regulators of Pol I (Grewal et al., 2005). However, the possible contribution of other nucleolar regulators in response to nutrient-dependent animal growth remains to be addressed. In addition to TIF-1A-dependent initiation, Pol I function is regulated at multiple levels. For example, Pol I elongation is facilitated by TFIIH, which also functions as a general transcription factor for Pol II (Assfalg et al., 2012; Iben et al., 2002). Several other factors, such as Nucleolin, Nucleophosmin, FACT, and Myb-binding protein 1a (MYBBP1A) have been shown to contribute to Pol I regulation, but their mechanism of action and regulation remains to be fully resolved (Birch et al., 2009; Hochstatter et al., 2012; Murano et al., 2008; Rickards et al., 2007). The rRNA-encoding genes are present in hundreds of repeats located in tandem in the genome (Grummt and Pikaard, 2003). rRNA expression is affected by the number of rRNA genes available for transcription (McStay and Grummt, 2008). This regulation occurs through epigenetic changes (McStay and Grummt, 2008); acetylation of histone H4 and tri-methylation of histone H3 lysine 4 correlates with a transcriptionally active rRNA gene, whereas methylation of histone H3 lysine 9 corresponds to a silent rRNA gene (Santoro and Grummt, 2005; Wang et al., 2007; Zhou et al., 2002). An important regulator of the epigenetic state of rRNA-encoding genes is the transcription termination factor TTF-I, which recruits Cockayne syndrome protein B (CSB) or nucleolar remodeling complex (NoRC) to activate or silence rDNA, respectively (Li et al., 2006; McStay and Grummt, 2008). Periodic tryptophan protein 1 (PWP1, also known as endonuclein) is a conserved WD40 domain-containing protein with poorly established function. Studies in mouse, Drosophila, and budding yeast (Saccharomyces cerevisiae) have shown that PWP1 associates with the chromatin and has a putative role in gene regulation (Casper et al., 2011; Suka et al., 2006). In Drosophila polytene chromosomes, PWP1 localization overlaps with that of active Pol II (Casper et al., 2011), and in mouse embryonic stem cells (mESCs), PWP1 association with chromatin correlates with high levels of H4K20me3 modification (Shen et al., 2015). PWP1 has recently been implicated in stem cell function. Loss of PWP1 function impairs the differentiation potential of mESCs (Shen et al., 2015), and Drosophila mutants lacking PWP1 display impaired male germline stem cell maintenance, leading to sterility (Casper et al., 2011). Therefore, the fly gene was named no child left behind. In budding yeast, PWP1 has been shown to associate with the chromatin of rDNA through interaction with the histone H4 tail (Suka et al., 2006), but its role in ribosomal gene regulation has not been explored further. Here, we uncover the essential role of PWP1 in nutrient-dependent control of animal growth. Mechanistically, PWP1 is regulated by mTORC1-mediated nutrient sensing via gene expression and phosphorylation, and it associates with Pol I to control nucleolar rRNA expression along with the epigenetic status of rRNA-encoding genes. Furthermore, we provide evidence that PWP1 expression is a critical determinant of tumor aggressive-

ness, suggesting that PWP1 is a potential target for cancer therapy. RESULTS PWP1 Regulates Tissue Growth The no child left behind (nclb) gene encodes the Drosophila ortholog of PWP1/endonuclein, a conserved WD40 repeat containing protein (36% amino acid identity between Drosophila and human, Figure S1A). We encountered dPWP1 upon searching for new growth-regulating genes; depletion of dPWP1 from the posterior compartment of the developing wing led to significantly reduced compartment size (Figures 1A and 1B) and modestly increased wing hair density, which is an indicator of reduced cell size (Figure 1C). Furthermore, dpwp1 mutant alleles, hypomorphic dpwp1nclb1, null allele dpwp1nclb2, and their transheterozygous combination dpwp1nclb1/2 (Casper et al., 2011) (Figure S1B) displayed strong larval undergrowth phenotypes (Figure S1C), while loss of dPWP1 had no effect on larval feeding activity (Figure S1D). dpwp1nclb2 null mutants died as small larvae, but hypomorphic dpwp1nclb1/2 mutants are semiviable and displayed delayed pupation kinetics and significantly reduced pupal volume (Figures 1D–1F). These are phenotypes commonly observed upon impaired growth signaling (Delanoue et al., 2010). To explore the role of dPWP1 in cell proliferation, we utilized dsRNA-mediated dPWP1 knockdown in Drosophila S2 cells. Depletion of dPWP1 prominently inhibited the proliferation of S2 cells (Figures 1G and S1E). Similarly, siRNA-mediated PWP1 knockdown inhibited the proliferation of human HeLa and U2OS cells (Figures 1H, S1F, S1G, and S1H). In sum, our data show that dPWP1 is a regulator of cell proliferation and tissue growth and suggest that it is functionally conserved in humans. PWP1 Regulates Ribosome Biogenesis In addition to small body size, the adult dpwp1nclb1/2 mutant flies had short and thin bristles (Figure 2A), which is a hallmark of the Minute phenotype caused by mutations impairing ribosome biogenesis or translation (Marygold et al., 2007). Earlier studies have shown that the inhibition of ribosome biogenesis in the fat body leads to systemic growth impairment (Delanoue et al., 2010; Marshall et al., 2012). Similarly, the depletion of dPWP1 in the fat body using the Cg-GAL4 driver led to delayed larval development (Figure 2B) and significantly reduced pupal volume (Figures 2C and 2D). Thus, in addition to tissue autonomous growth control, dPWP1 regulates animal growth systemically via its function in the fat body. PWP1 has a conserved nucleolar localization sequence (NoLS; predicted by NoD, Figure S1A) (Scott et al., 2011). By using the specific dPWP1 antibody (Casper et al., 2011; Figure S2A), we explored the subcellular localization of endogenous dPWP1 in the fat-body cells, which allow good spatial resolution and are critically involved in organismal growth control (Colombani et al., 2003). In the early third instar larvae, dPWP1 was localized in the nucleoplasm and in the middle of the nucleoli and was adjacent to the strongly fibrillarin positive ‘‘dense fibrillar components’’ (Figure 2E and Movie S1) (Goessens et al., 1987; Hernandez-Verdun, 2006; Huang, 2002). Developmental Cell 43, 240–252, October 23, 2017 241

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Figure 1. PWP1 Regulates Tissue Growth and Proliferation (A) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) leads to reduced compartment size. (B) Quantification of the ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas in (A). (C) Cell density ratio between posterior (P) (n = 10) and anterior (A) (n = 10) compartments in (A). (D) Pupation kinetics of control (n = 5) and dpwp1nclb1/2 (n = 5) larvae. dAEL, days after egg laying. (E) Representative images of control and dpwp1nclb1/2 pupae. (F) Quantification of pupal volumes of control (n = 4) and dpwp1nclb1/2 (n = 4) pupae. (G) Proliferation in Ctrl (Lac dsRNA) (n = 3) and dPWP1-specific dsRNA (n = 3) treated S2 cells. (H) Proliferation of HeLa cells after transfection with non-targeting (Ctrl) (n = 3) or PWP1-specific (n = 3) siRNAs. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S1.

To directly assess the role of PWP1 in ribosome biogenesis, we analyzed Pol I-dependent rRNA expression. Indeed, the levels of 5.8S, 18S, and 28S rRNA species were significantly downregulated in the dpwp1 mutants, dpwp1nclb2 null mutants displaying the most prominent reduction of rRNA expression (Figure 2F). Similar to Drosophila, depletion of PWP1 from HeLa cells led to reduced rRNA levels (Figure S2B). Ribosomal gene expression requires the activity of all three RNA polymerases. Therefore, we tested whether ribosomal targets of Pol II and Pol III displayed differential expression following the loss of PWP1. While the expression of Pol II-dependent ribosome biogenesis genes was unchanged or moderately upregulated in Drosophila mutants lacking dPWP1 and in HeLa cells following PWP1 knockdown (Figure S2C and S2D), the expression of Pol III-dependent 5S rRNA, 7SLRNA, and tRNAs was significantly downregulated in dpwp1 mutants (Figure S2E). Thus, PWP1 affects the levels of rRNA species transcribed by Pol I and Pol III. PWP1 Associates with rDNA Chromatin and Promotes Pol I-Mediated Transcription To get a more mechanistic understanding on the role of PWP1, we further addressed its nucleolar function. Immunofluorescence analysis in HeLa cells revealed prominent nucleolar localization of human PWP1 and colocalization with the Pol I subunit POLR1E (Figures 3A and S3A). Furthermore, co-immunoprecipitation experiments with POLR1E antibodies suggested that PWP1 is present in a complex with Pol I (Figure 3B). This was 242 Developmental Cell 43, 240–252, October 23, 2017

further corroborated by chromatin immunoprecipitation, which revealed PWP1 association with the rDNA region as well as with the rDNA promoter (Figure 3C), while no association between PWP1 and Pol II-dependent ribosome biogenesis genes was detected (Figure S3B). Since there was no Drosophila Pol I antibody available, we analyzed the localization of Cdk7, a subunit of a general transcription factor TFIIH, which has been shown to associate with Pol I (Assfalg et al., 2012). Fat-body cells displayed an enrichment of Cdk7 in the central region of the nucleolus, which localized in the close vicinity to dPWP1 (Figure 3D). In conclusion, PWP1 acts in close contact with Pol I at the rDNA chromatin. To more directly assess the possible involvement of PWP1 in rRNA transcription, we analyzed the synthesis of new rRNA in human U2OS cells by 4-thiouridine (4sU) labeling. Consistent with the idea that PWP1 is a positive regulator of Pol I function, PWP1 knockdown led to reduced levels of newly synthetized rRNA (Figure 3E). One regulatory level of the rDNA locus is through histone modifications, which determine how many of the rDNA loci are accessible for transcription (McStay and Grummt, 2008). Intriguingly, siRNA-mediated depletion of PWP1 in U2OS cells led to a marked decrease of histone H4 lysine 12 (H4K12) acetylation, which is an activating modification (Figure 3F). Furthermore, levels of silencing H3K9 dimethylation were increased upon PWP1 knockdown (Figure 3G). In conclusion, our data show that PWP1 promotes Pol I-mediated transcription and regulates the epigenetic status of rDNA.

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Figure 2. PWP1 Regulates Pol I-Mediated Ribosomal Gene Expression (A) dpwp1nclb1/2 mutants display short and thin bristles. (B) Pupation kinetics of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted larvae. dAEL, days after egg laying. (C) Representative images of control and dPWP1 fat-body (Cg-Gal4)-depleted pupae. (D) Quantification of volumes of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted pupae. (E) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with fibrillarin in fat bodies of early third instar larvae. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, and 28S rRNA (RNA polymerase I targets) expression in control larvae (n = 3) and dpwp1 mutants (n = 3). cdk7 was used as a reference gene. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S2.

PWP1 Functionally Cooperates with MYBBP1A and Nucleolin To get a comprehensive view on the PWP1-associated proteins, we performed PWP1 affinity purifications from both Drosophila S2 cells and human HEK293 Flp-In TREx cells, followed by mass spectrometry (AP-MS). The human HEK293 Flp-In TREx cell system allowed us to express tagged PWP1 at the endogenous level (Figure S4A). In both settings, PWP1 co-purified with a number of TRiC/CCT chaperonin subunits (Figures S4B and S4C; Tables S1A and S1B). This was consistent with the fact that PWP1 contains WD40 repeats, which are known substrates of the TRiC/CCT chaperonins (Miyata et al., 2014; Yam et al., 2008). In agreement with our earlier data, the AP-MS analyses captured a number of nucleolar interaction partners, such as BRX1, fibrillarin, and Nop56 (Figures 4A and 4B). These proteins have established functions in pre-rRNA processing and early ribosomal maturation (Gautier et al., 1997; Kaser et al., 2001; Tollervey et al., 1993). A particularly interesting human PWP1 interaction partner was Myb-binding protein 1A (MYBBP1A), which has been previously suggested to coordinate Pol I-mediated transcription and rRNA processing (Hochstatter et al., 2012; Tan et al., 2012). We

confirmed the conservation of the interaction between PWP1 and MYBBP1A in Drosophila by co-immunoprecipitation in S2 cells (Figure 4C). To complement the PWP1 affinity purification analysis, we also performed a BioID analysis of PWP1-associated proteins in human HEK293 cells. BioID is based on the activity of a promiscuous BirA biotin ligase, which labels the proteins that are in close proximity with the BirA-tagged bait (Roux et al., 2001). This differs from affinity purification of protein complexes, since it allows identification of highly transient interactions. Interestingly, the BioID strategy led to identification of Nucleolin, a nucleolar protein with an established role in rRNA transcription and processing (Cong et al., 2012; Ginisty et al., 1998; Table S1C). Moreover, immunofluorescence analysis revealed nucleolar colocalization between PWP1 and Nucleolin (Figure 4D), and depletion of PWP1 in U2OS cells led to striking re-localization of Nucleolin, including increased nucleoplasmic localization (Figure 4E). In conclusion, PWP1 associates with known regulators of rRNA expression, including MYBBP1A and Nucleolin. Next, we wanted to functionally analyze the roles of MYBBP1A and Nucleolin with respect to PWP1 function. As the in vivo role of MYBBP1A is poorly established, we generated mutants of Developmental Cell 43, 240–252, October 23, 2017 243

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Figure 3. PWP1 Associates with rDNA Chromatin and Promotes Pol I-Mediated Transcription (A) Representative immunofluorescent images of PWP1 and POLR1E localization in HeLa cells. Scale bar, 5 mm. (B) Co-purification of GFP-tagged PWP1 upon pull-down of V5-tagged POLR1E from HeLa cells. Tubulin serves as a loading control. (C) Chromatin immunoprecipitation (ChIP) revealed the enrichment of PWP1 binding across an rDNA repeat unit and promoter (n = 3). (D) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with dCDK7 in fat bodies of early third instar larvae. Scale bar, 5 mm. (E) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green staining) and newly transcribed RNA (upper panel, visualized by streptavidin-horseradish peroxidase [HRP] detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. (F) ChIP revealed the relative level of H4K12Ac on different regions of rDNA in U2OS cells following PWP1 depletion. Ratio of 1 between control and PWP1

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Drosophila MYBBP1A (lethal [1] 1Bi) by CRISPR/Cas9 (Figure S4D). Animals lacking functional MYBBP1A displayed low expression of rRNA (Figure 4F) along with strong larval undergrowth (Figure 4G), implying that MYBBPIA is needed for full rRNA expression in vivo in Drosophila. Furthermore, fat-bodyspecific knockdown of MYBBP1A led to dissociation of nucleolar dPWP1 (Figures 4H and 4I). Earlier studies on mammalian MYBBP1A have shown that it is a negative regulator of rRNA transcription while promoting pre-rRNA processing (Hochstatter et al., 2012). Nucleolin also regulates several steps of ribosome biogenesis. Earlier data on the role of Nucleolin in rRNA transcription is conflicting, with evidence supporting both positive and negative regulation (Durut and Sa´ez-Va´squez, 2015). To directly measure the new rRNA synthesis, we utilized 4sU labeling in U2OS cells. Consistent with the earlier findings, siRNAmediated depletion of MYBBP1A led to increased 4sU labeling of rRNA (Figure 4J). Interestingly, we also observed that knockdown of Nucleolin led to increased 4sU labeling, implying that in this experimental setting, Nucleolin represses rRNA transcription (Figure 4J). The increased 4sU labeling allowed us to perform an epistasis experiment with PWP1 and its interaction partners. Intriguingly, knockdown of PWP1 fully prevented the increased 4sU labeling of rRNA caused by knockdown of MYBBP1A and Nucleolin (Figure 4K). In conclusion, PWP1 displays complex functional cooperation with MYBBP1A and Nucleolin. mTORC1-Dependent Phosphorylation Regulates Nucleolar Localization of PWP1 Tissue growth and ribosome biogenesis are under the control of nutrient-regulated signaling pathways, including the insulin/ mTOR pathway (Hietakangas and Cohen, 2009). To explore the possible involvement of dPWP1 in nutrient-dependent signaling, we analyzed the nucleolar localization of dPWP1 in the Drosophila fat body under different dietary regimes. Intriguingly, acute starvation of early third instar larvae led to dissociation of nucleolar dPWP1 (Figures 5A and 5B), which was restored upon re-feeding (Figures 5C and 5D). This implies that the nucleolar function of dPWP1 is regulated with respect to the nutrient status of the animal. Accordingly, nucleolar dPWP1 dissociated at the wandering stage, during which the animals stop feeding (Figure S5A). To test whether mTORC1 signaling controls dPWP1 localization, we fed the larvae with rapamycin. Similar to starvation, rapamycin feeding led to dissociation of nucleolar dPWP1, demonstrating that dPWP1 localization is controlled by mTORC1 signaling (Figures 5E and 5F). The mTORC1-dependent regulation of nucleolar dPWP1 led us to explore possible dPWP1 phosphorylation by using the Phos-tag SDS-PAGE (Kinoshita et al., 2006). V5-tagged dPWP1 transfected in S2 cells displayed several slow migrating bands, which were abolished by phosphatase treatment (Figures 5G and S5B), confirming that they were reflecting dPWP1 phosphorylation. Intriguingly, most of the bands representing

siRNA-treated samples (expected when no change occurs) is indicated by a dashed line. (n = 3). (G) ChIP revealed the relative level of H3K9me2 on different regions of rDNA in U2OS following PWP1 depletion (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S3.

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Figure 4. PWP1 Functionally Cooperates with MYBBP1A and Nucleolin (A) Summary of dPWP1 interacting ribosome biogenesis regulators in S2 cells. (B) Summary of PWP1 interacting ribosome biogenesis regulators in HEK293 cells. (C) Co-purification of HA-tagged dPWP1 with dMYBBP1A upon pull-down of V5-tagged dMYBBP1A from S2 cells. Tubulin serves as a loading control. (D) Representative immunofluorescent images of PWP1 and Nucleolin localization in U2OS cells. Scale bar, 5 mm. (E) Representative immunofluorescent images of Nucleolin localization in U2OS cells followed by PWP1 depletion. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, 28S, and 5S rRNA expression in control larvae (n = 3) and dmybbp1a mutants (n = 3). cdk7 was used as a reference gene. (G) Representative images of control (w-) and mybbp1a mutant larvae at 96 hr after egg laying. (H) Representative immunofluorescent images of dPWP1 localization in fat bodies of early third instar larvae. Depletion of dMYBBP1A from the fat body (Fb-GAL4) leads to the dissociation of nucleolar dPWP1. Scale bar, 5 mm. (I) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (H). A total of 15 nuclei from 3 independent fat bodies were quantified. (J and K) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green or ethidium bromide staining) and newly transcribed RNA (upper panel, visualized by streptavidin-HRP detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min of 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S4.

phosphorylated dPWP1 were induced by insulin treatment and were strongly inhibited by rapamycin (Figure 5G), consistent with mTORC1-dependent dPWP1 phosphorylation. The

mTORC1 pathway controls the activity of other kinases, including S6 kinase (S6K) (Burnett et al., 1998). We tested the possible involvement of S6K in dPWP1 phosphorylation. While Developmental Cell 43, 240–252, October 23, 2017 245

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(A) Representative immunofluorescent images of endogenous dPWP1 localization in fat bodies of early third instar fed and starved (6 hr) larvae. Scale bar, 5 mm. (B) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (A). A total of 15 nuclei from 3 independent fat bodies were quantified. (C) Representative immunofluorescent images of endogenous dPWP1 localization in fat bodies of early third instar starved (4 hr) and re-fed (6 hr) larvae. Scale bar, 5 mm. (D) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (C). A total of 15 nuclei from 3 independent fat bodies were quantified. (E) Representative immunofluorescent images of dPWP1 localization in fat bodies dissected from early third instar larvae fed without or with rapamycin (14 hr). Scale bar, 5 mm. (F) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (E). A total of 15 nuclei from 3 independent fat bodies were quantified. (G) Immunoblot of S2 cell lysates expressing a V5-tagged form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin alone (10 min) or in combination with rapamycin (2 hr). Phospho-dPWP1 species (anti-V5) are indicated by arrowheads. (H) Immunoblot of lysates of S2 cell expressing a V5-tagged form of dPWP1 together with RNAi against LacZ (ctrl), dS6K, or dRaptor resolved on Phos-tag SDS-PAGE. Cells were treated without or with insulin (10 min). (I) Quantification of phospho-dPWP1 species of (H). (J) Immunoblot of S2 cell lysates expressing a V5-tagged wild-type or S384 alanine mutated form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin (10 min). (K) Quantification of phospho-dPWP1 species of (J). (L) Immunofluorescent analysis of fat body expressing the wild-type and S384A form of dPWP1 dissected from early third instar larvae. Scale bar, 5 mm. (M) Quantification of the nucleolus/nucleoplasm localization ratio in (L). A total of 15 nuclei from 3 independent fat bodies were quantified. **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S5.

depletion of Raptor (an essential subunit of mTORC1) abolished the phosphorylation of dPWP1, knockdown of S6K had no detectable effect (Figures 5H, 5I, S5C, and S5D). Thus, mTORC1 likely controls dPWP1 phosphorylation independently of S6K. In order to map the target site(s) of dPWP1 phosphorylation, we systematically mutated all serines and threonines into alanines. While mutation of most amino acids had no significant effect on dPWP1, S251A mutation reduced the expression level of dPWP1, but did not affect phosphorylation, whereas S384A mutation abolished dPWP1 phosphorylation (Figures 5J, 5K, S5E, and S5F), suggesting S384 is a key target site of PWP1 phosphorylation. To directly explore whether mTORC1-dependent phosphorylation regulates the nucleolar localization of dPWP1, we generated transgenic flies harboring wild-type and phosphorylation-deficient S384A mutant forms of dPWP1 and expressed them in the fat body. Whereas wild-type dPWP1 displayed similar subcellular localization with endogenous dPWP1, S384A mutation significantly reduced the nucleolar localization of dPWP1 (Figures 5L and 5M). We also tested the localization of a putative phosphomimetic S384E mutant, but it localized poorly into the nucleolus (Figure S5G), implying that an intact phosphorylation site is needed for proper nucleolar localization of PWP1. In conclusion, nucleolar localization of dPWP1 is regulated by mTORC1-dependent phosphorylation.

dpwp1 Gene Expression Is Under the Control of the Insulin/mTOR/Myc Pathway In addition to phosphorylation, dpwp1 gene expression is regulated through nutrient-responsive pathways. dpwp1 mRNA levels were prominently increased when starved animals were re-fed with a protein-rich yeast diet but was not comparatively affected by a high-sugar diet (Figure 6A). Because mTORC1 is regulated by cellular amino acid levels (Hietakangas and Cohen, 2009), we explored whether the dietary protein-induced expression of dpwp1 was mTOR dependent. Indeed, the induction of dpwp1 expression upon yeast feeding was blunted in mTOR mutant larvae (Figure 6B), implying that the elevated dPWP1 expression by dietary protein is under the control of mTOR signaling. In line with this, activation of mTORC1 signaling by overexpressing Insulinlike receptor (InR) or Ras homolog enriched in brain (Rheb) increased dpwp1 expression (Figures 6C and 6D). One established downstream effector of mTOR signaling is the transcription factor Myc, which is known to control ribosome biogenesis (Teleman et al., 2008). We tested if dpwp1 expression is regulated in a Myc-dependent manner and observed reduced dpwp1 expression upon Myc knockdown and elevated dpwp1 expression upon Myc overexpression (Figure 6E). The data are consistent with the model that Myc acts downstream of mTOR signaling to positively regulate dpwp1 gene expression. dPWP1 Is Functionally Downstream of NutrientResponsive Signaling To functionally test the possible involvement of dPWP1 in tissue growth driven by insulin/mTORC1 signaling and Myc, we performed genetic epistasis experiments with dpwp1, InR, Rheb, and myc in the developing wing. As previously shown (Johnston et al., 1999; Saucedo et al., 2003; Tiefenbo¨ck et al., 2010; Werz et al., 2009), overexpression of InR, Rheb, and Myc led to significant tissue overgrowth (Figures 6F, 6G, S6A, and S6B). However, when dPWP1 was simultaneously depleted by RNAi, InR, Rheb, and Myc failed to promote growth (Figures 6F, 6G, S6A, and S6B), consistent with the idea that dPWP1 acts genetically downstream of the insulin/mTORC1/Myc pathway to regulate tissue growth. To get more mechanistic insight into the dPWP1 function downstream of mTOR signaling, we analyzed Cdk7 localization, which marks the TFIIH complex, an essential elongation factor of Pol I (Iben et al., 2002; Assfalg et al., 2012). Depletion of dPWP1 by RNAi reduced the levels of Cdk7 in the whole nucleus, including the nucleolus (Figure 6H). However, when dPWP1 was depleted simultaneously with the activation of mTORC1 (Rheb overexpression), a striking re-localization of Cdk7 was observed. Cdk7 displayed diffuse nucleolar localization, except for the very center of the nucleolus, which remained devoid of Cdk7 (Figure 6H). Thus, dPWP1 cooperates with mTORC1 signaling to control nucleolar TFIIH in vivo. The insulin/mTOR-mediated dPWP1 regulation and the genetic epistasis experiments imply that dPWP1 has a key role in nutrient-dependent regulation of Pol I function. This was confirmed by the finding that dpwp1nclb2 mutant larvae failed to increase rRNA expression upon re-feeding, in contrast to control animals (Figure 6I).

High PWP1 Expression Predicts Poor Outcome of Head and Neck Squamous Cell Carcinoma The important role for PWP1 in mediating growth and rRNA expression downstream of mTOR indicate that it may have a role in cancer cells with constitutively high mTOR signaling activity. Head and neck squamous cell carcinoma (HNSCC) has recently been identified as a cancer type with particularly strong evidence for hyperactivation of the mTOR pathway (Lui et al., 2013), and the functional interaction partner for PWP1, MYBBP1A, was recently shown to promote proliferation of HNSCC cells (Sanhueza et al., 2012). However, neither a functional nor clinical role of PWP1 in HNSCC, nor any other human cancer, has been studied as yet. To this end, we analyzed the possible impact of PWP1 and its functional interaction partners, Nucleolin and MYBBP1A, on the proliferation of patient-derived HNSCC cells and observed significantly inhibited proliferation upon depletion of each protein (Figures 7A and 7B). To analyze the expression of PWP1 in tumor samples derived from HNSCC patients, we performed IHC staining on whole HNSCC tissue € et al., sections from 69 patients (Routila et al., 2016; Ventela 2015). Most of the tumors displayed clear PWP1 positive staining, while the surrounding normal tissue was only marginally stained with PWP1 antibodies (Figure 7C, Table S2). When samples were divided into two groups according to the level of PWP1 expression (low versus high), a strong correlation between PWP1 expression levels and patient mortality was observed (Figure 7D), implying that PWP1 expression is an important determinant of HNSCC aggressiveness and a marker for poor prognosis. DISCUSSION Nucleolar regulation of ribosome biogenesis is an important hub for animal growth control. How the nucleolar functions are dynamically regulated upon developmental growth and how the information about animal nutrient status is mediated by nutrient-sensing pathways to control nucleolar regulatory proteins have remained insufficiently understood. Here, we establish PWP1 as a regulator of tissue growth, which acts in the interface between nutrient-sensing mTORC1 signaling and the growth rate determining rRNA expression in the nucleolus. Specifically, we demonstrate that (1) PWP1 positively regulates Pol I-dependent rRNA expression and consequently facilitates tissue growth; (2) PWP1 associates with Pol I and maintains the chromatin of rDNA in transcription-competent state; (3) PWP1 functionally cooperates with other regulators of Pol I activity, including MYBBP1A and Nucleolin; (4) PWP1 is regulated by mTORC1 through gene expression and phosphorylation, which regulates the nucleolar localization of PWP1; (5) PWP1 synergizes with mTOR signaling to control the nucleolar localization of TFIIH, an elongation factor of Pol I; (6) PWP1 is essential for proliferation of cancer cells, including patientderived HNSCC cells, and high PWP1 expression in HNSCC tumors correlates with poor survival of patients. We observed that PWP1 is an essential regulator of Pol I downstream of mTOR signaling in vivo. Previous studies in Drosophila have established that nucleolar ribosome biogenesis is under the control of mTOR signaling. For example, elevated mTOR activity leads to increased nucleolar volume in the wing imaginal disc, and loss of mTOR activity halts the expression of pre-rRNA in Developmental Cell 43, 240–252, October 23, 2017 247

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Figure 6. dPWP1 Is Involved in Nutrient-Dependent Growth Control (A) qRT-PCR analysis of dPWP1 mRNA expression in third instar larvae upon starvation (24 hr) (n = 3) or re-feeding with a high-protein diet (20% yeast, 6 hr) (n = 3) or a high-sugar diet (20% sucrose, 6 hr) (n = 3). cdk7 was used as a reference gene.

248 Developmental Cell 43, 240–252, October 23, 2017

the larvae (Grewal et al., 2007). The general transcription factor TIF-IA is a known target for mTOR in yeast and mammals (Claypool et al., 2004; Mayer et al., 2004), and in Drosophila, it is essential for mTOR-dependent growth (Grewal et al., 2007). Notably, however, mTOR-dependent phosphorylation of TIF-IA remains to be established in Drosophila. Here, we demonstrated mTORdependent phosphorylation of dPWP1 and provided functional evidence for the importance of this phosphorylation in the nucleolar localization of dPWP1. This suggests that mTOR is likely to regulate nucleolar rRNA expression through phosphorylation of multiple targets. Earlier studies in Drosophila have also shown that TIF-IA expression is nutrient dependent (Ghosh et al., 2014) and positively regulated by transcription factor Myc (Grewal et al., 2005). This parallels with our data on the regulation of dpwp1 gene expression, which is also nutrient and Myc responsive. Similar to earlier findings showing TIF-IA to be essential for the growth-promoting function of Myc (Grewal et al., 2005), we observed that dPWP1 knockdown was sufficient to prevent overgrowth caused by Myc overexpression. Multicellular animals coordinate their growth through cell and tissue autonomous mechanisms as well as through hormonal mechanisms, which provide growth coordination at the level of the whole animal. We observed that dPWP1 controls growth at both levels. In the wing imaginal disc, dPWP1 is essential for tissue autonomous growth, while inhibition of dPWP1 in the fat body leads to systemic inhibition of growth, reflected as delayed larval development and reduced body size. This is in line with earlier observations on fat-body-specific inhibition of mTORC1 as well as reduction of amino acid intake (Colombani et al., 2003). Recent studies have shed new light into the fat-bodymediated systemic growth control. mTOR signaling in the fat

(B) qRT-PCR analysis of dPWP1 mRNA expression in control (w ) (n = 3) and TOR mutant (TORdeIP) (n = 3) second instar larvae on 5% sucrose food (48 hr) and 20% yeast food re-feeding (20 hr). All samples were normalized to the 5% sugar-starved control larvae. cdk7 was used as a reference gene. (C) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and InR overexpressing (n = 3) second instar larvae (48 hr). cdk7 was used as a reference gene. (D) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and Rheb overexpressing (n = 3) second instar larvae (48 hr). rp49 was used as a reference gene. (E) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and Myc RNAi or overexpressing (n = 3) second instar larvae (48 hr). cdk7 was used as a reference gene. (F) dPWP1 RNAi in the posterior compartment of the developing wing (EnGal4) alone or in combination with insulin-like receptor (InR) overexpression. The ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas was quantified. (G) dPWP1 RNAi in the posterior compartment of the developing wing (EnGal4) alone or in combination with Rheb or Myc overexpression. The ratio of posterior (P) (n = 12) and anterior (A) (n = 12) wing areas was quantified. (H) Immunofluorescence analysis shows dCDK7 localization in the fat bodies of early third instar larvae with dPWP1 RNAi alone or combination with Rheb overexpression. Scale bar, 5 mm. (I) qRT-PCR analysis of rRNA expression upon yeast re-feeding of control (n = 3) and dpwp1nclb2 null mutant (n = 3) larvae. cdk7 was used as a reference gene. ANOVA showed a significant genotype by feeding interaction in terms of 18S and 28S rRNA expression (5.8S: F1, 8 = 1.44, p = 0.26; 18S: F1, 8 = 7.92, p = 0.02; 28S: F1, 8 = 8.41, p = 0.02). *p < 0.05, **p < 0.01, ***p < 0.001 (I was analyzed by ANOVA, all others by Student’s t test). Error bars indicate SDs. See also Figure S6.

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Figure 7. PWP1 Is Highly Expressed in Aggressive HNSCC Tumors (A) Proliferation of control (n = 3) and PWP1 (n = 3)- or MYBBP1A (n = 3)depleted HNSCC (UT-SCC-2) cells. (B) Proliferation of control (n = 3) and Nucleolin (n = 3)-depleted HNSCC (UT-SCC-2) cells. (C) Representative immunohistochemistry images of HNSCC tissue sections displaying weak and strong PWP1 expression. Normal tissue devoid of PWP1 signal is indicated by arrowheads. Scale bar, 100 mm. (D) Kaplan-Meier survival curves of patients with HNSCC stratified for weak and strong PWP1 expression. Error bars indicate SDs.

body affects the release of hormonal signals, such as Growth Blocking Peptides (GBPs) 1 and 2, TNF-a Eiger as well as Stunted (Agrawal et al., 2016; Delanoue et al., 2016; Koyama and Mirth, 2016). These hormones are relayed to the insulin-producing cells (IPCs), providing systemic growth control through the secretion and expression of insulin-like peptides (ILPs). It remains possible that dPWP1 directly regulates the expression of fat-body-derived hormones or alternatively, there is a common surveillance mechanism, which responds to changes of ribosome biogenesis in the fat body. In fact, such mechanism exists in the Drosophila IPCs, where inhibited ribosome biogenesis leads to activation of an atypical MAP kinase ERK7, which prevents ILP secretion (Hasygar and Hietakangas, 2014). While our study focused mainly on PWP1 function in the nucleolus, it should be noted that a large fraction of PWP1 in the fat body is localized in the nucleoplasm. Our data also suggests that dPWP1 might regulate ribosome biogenesis through Pol III activity as well (Figure S2E). Thus, PWP1 is a candidate to provide coordination between the two RNA polymerases responsible for rRNA expression. Since mTORC1 also controls Pol III (Kantidakis and White, 2010; Marshall et al., 2012; Wei et al., 2009), it remains an interesting possibility that PWP1 is under mTORC1 control also in this setting. Considering the prominent synergistic effects of dPWP1 and mTOR activation on Cdk7 localization in vivo, it will be interesting to explore whether altered TFIIH function contributes to the gene expression by all three RNA polymerases in response to nutrient-induced signaling. Furthermore, TORC1 activity is essential for proper rRNA processing, but the underlying mechanisms remain to be elucidated (Iadevaia et al., 2012). Association with proteins involved in rRNA processing implies that PWP1 might also be involved in coordinating the co-transcriptional processing of rRNAs. Similar roles have been shown for the PWP1-associated proteins MYBBP1A and Nucleolin (Ginisty et al., 1998; Hochstatter et al., 2012). Furthermore, we observed that in mammalian cells, PWP1 contributes to the epigenetic status of rRNA-encoding genes. Earlier observations from mouse embryonic stem cells have shown that PWP1 chromatin association overlapped with high levels of H4K20me3 modification (Shen et al., 2015), further supporting the idea that PWP1 is functionally coupled to epigenetic regulation and suggesting that the epigenetic role of PWP1 expands beyond its role in rRNA regulation. Upregulation of ribosome biogenesis is frequently observed in malignant tumors and is likely playing an important role in driving tumor growth (Orsolic et al., 2016). Moreover, the p53-mediated nucleolar stress response, which halts cell proliferation and triggers apoptosis upon impaired nucleolar ribosome biogenesis, provides possible means to inhibit cancer growth (Woods et al., 2015). Therefore, there is increasing interest toward strategies to target the nucleolar functions of cancer cells. Earlier studies have shown that Nucleolin, a nucleolar protein functionally interacting with PWP1, is highly expressed in tumor cells (Durut and Sa´ez-Va´squez, 2015). Moreover, the binding partner of PWP1, MYBBP1A, has been shown to promote proliferation of HNSCC cells (Sanhueza et al., 2012). Intriguingly, depletion of PWP1 (along with that of MYBBP1A and Nucleolin) impaired the proliferation of patient-derived HNSCC cells. We also observed high PWP1 expression in HNSCC samples and a clear inverse correlation between PWP1 expression level and patient Developmental Cell 43, 240–252, October 23, 2017 249

survival. These findings imply that PWP1 expression might create a vulnerability in HNSCC tumors. As expression of PWP1 in the surrounding normal tissue was very low, PWP1 could constitute an interesting drug target for cancer therapies. PWP1 possesses a WD40 repeat domain, and recent reports on small inhibitory molecules binding to WD40 domains suggest that PWP1 may indeed be a druggable target (Guillermo et al., 2013; Orlicky et al., 2010). However, to gain a comprehensive view on the therapeutic potential of PWP1 inhibition, a systematic survey on PWP1 expression and the functional role in human tumors is certainly warranted. In conclusion, our study establishes PWP1 as a novel regulator of developmental growth through the control of Pol I-mediated rRNA transcription. Furthermore our findings provide evidence for the mechanism and functional consequences of PWP1 regulation by the mTOR signaling pathway, implying that nutrientresponsive regulation of nucleolar functions are more elaborate than previously anticipated. The association between PWP1 expression and HCSCC aggressiveness suggest that PWP1 may have therapeutic or diagnostic potential in the clinic. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Lines B Drosophila Stocks B Patient Samples METHOD DETAILS B Plasmids, RNAi and Cell Culture B Drosophila Genetics B Food Consumption Assay B Growth Phenotype Analyses B Quantitative RT-PCR B Western Blot and Immunostainings B Alanine Scanning B Co-Immunoprecipitations and Immunoblotting B Chromatin Immunoprecipitation B Single-Step Affinity Purification B BioID B Mass Spectrometry B 4sU-Labeling of RNA B Immunohistochemistry QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

the AP-MS and BioID analyses and wrote the corresponding parts of the manuscript. S.V., J.S., O.K., R.G., and J.W. analyzed HNSCC tumors and contributed patient-derived HNSCC cells. N.L. analyzed the regulation of dpwp1 expression by Rheb/Myc and feeding activity of pwp1 mutants. W.Z. generated Mybbp1a mutant fly and analyzed its growth phenotype. Y.L., J.M., J.W., and V.H. wrote the manuscript.

ACKNOWLEDGMENTS We thank Mark van Doren, Beat Suter, and Marikki Laiho for fly stocks, antibodies, reagents, and cells. Mikko Frilander, Bhupendra Verma, Heini € la €, Eiji Kinoshita, Yan Yan, Heini Hakala, Ying Yang, and Wei Ting as Seppa well as the Genome Biology Unit and the Light Microscopy Unit of the Institute of Biotechnology are thanked for technical help and advice. Richard Melvin is thanked for help with statistics and the other members of the Hietakangas group for feedback. This research was funded by the Academy of Finland (grant nos. 263756 and 286767 to V.H.), European Research Council (grant no. 281720 to V.H.), Sigrid Juse´lius Foundation (to V.H.), Novo Nordisk Foundation (NNF16OC0021460 to V.H.), Helsinki Institute of Life Science (to V.H.), and the Integrative Life Science doctoral program (to Y.L.). Received: June 16, 2016 Revised: August 1, 2017 Accepted: September 25, 2017 Published: October 23, 2017 REFERENCES Agrawal, N., Delanoue, R., Mauri, A., Basco, D., Pasco, M., Thorens, B., and Le´opold, P. (2016). The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response. Cell Metab. 23, 675–684. Assfalg, R., Lebedev, A., Gonzalez, O.G., Schelling, A., Koch, S., and Iben, S. (2012). TFIIH is an elongation factor of RNA polymerase I. Nucleic Acids Res. 40, 650–659. Birch, J.L., Tan, B.C.-M., Panov, K.I., Panova, T.B., Andersen, J.S., OwenHughes, T.A., Russell, J., Lee, S.-C., and Zomerdijk, J.C.B.M. (2009). FACT facilitates chromatin transcription by RNA polymerases I and III. EMBO J. 28, 854–865. Burnett, P.E., Barrow, R.K., Cohen, N.A., Snyder, S.H., and Sabatini, D.M. (1998). RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA 95, 1432–1437. Bywater, M.J., Pearson, R.B., McArthur, G.A., and Hannan, R.D. (2013). Dysregulation of the basal RNA polymerase transcription apparatus in cancer. Nat. Rev. Cancer 13, 299–314. Casper, A.L., Baxter, K., and Doren, M.V. (2011). No child left behind encodes a novel chromatin factor required for germline stem cell maintenance in males but not females. Development 138, 3357–3366. Claypool, J.A., French, S.L., Johzuka, K., Eliason, K., Vu, L., Dodd, J.A., Beyer, A.L., and Nomura, M. (2004). Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes. Mol. Biol. Cell 15, 946–956. Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J., and Le´opold, P. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114, 739–749.

Supplemental Information includes six figures, three tables, and one movie and can be found with this article online at https://doi.org/10.1016/j.devcel. 2017.09.022.

Cong, R., Das, S., Ugrinova, I., Kumar, S., Mongelard, F., Wong, J., and Bouvet, P. (2012). Interaction of nucleolin with ribosomal RNA genes and its role in RNA polymerase I transcription. Nucleic Acids Res. 40, 9441–9454.

AUTHOR CONTRIBUTIONS

Delanoue, R., Slaidina, M., and Le´opold, P. (2010). The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells. Dev. Cell 18, 1012–1021.

Y.L., J.M., and V.H. conceived the study, designed the experiments, and analyzed the data of most experiments. Y.L. performed most of the experiments to analyze PWP1 function and regulation. L.Y. and M.V. performed

Delanoue, R., Meschi, E., Agrawal, N., Mauri, A., Tsatskis, Y., McNeill, H., and Le´opold, P. (2016). Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor. Science 353, 1553–1556.

250 Developmental Cell 43, 240–252, October 23, 2017

Drygin, D., Rice, W.G., and Grummt, I. (2010). The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu. Rev. Pharmacol. Toxicol. 50, 131–156. Durut, N., and Sa´ez-Va´squez, J. (2015). Nucleolin: dual roles in rDNA chromatin transcription. Gene 556, 7–12. €dle, B., Friedel, C.C., Zimmer, R., Mages, J., Do¨lken, L., Ruzsics, Z., Ra Hoffmann, R., Dickinson, P., Forster, T., Ghazal, P., et al. (2008). High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–1972. Fietz, M.J., Jacinto, A., Taylor, A.M., Alexandre, C., and Ingham, P.W. (1995). Secretion of the amino-terminal fragment of the Hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila. Curr. Biol. 5, 643–650. Gautier, T., Berges, T., Tollervey, D., and Hurt, E. (1997). Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis. Mol. Cell. Biol. 17, 7088–7098. Gentilella, A., Kozma, S.C., and Thomas, G. (2015). A liaison between mTOR signaling, ribosome biogenesis and cancer. Biochim. Biophys. Acta 1849, 812–820. Ghosh, A., Rideout, E.J., and Grewal, S.S. (2014). TIF-IA-dependent regulation of ribosome synthesis in Drosophila muscle is required to maintain systemic insulin signaling and larval growth. PLoS Genet. 10, e1004750. Ginisty, H., Amalric, F., and Bouvet, P. (1998). Nucleolin functions in the first step of ribosomal RNA processing. EMBO J. 17, 1476–1486. Goessens, G., Thiry, M., and Lepoint, A. (1987). Relations between Nucleoli and Nucleolus-organizing Regions during the Cell Cycle (Springer), pp. 261–271. Gre´nman, R., Carey, T.E., McClatchey, K.D., Wagner, J.G., Pekkola-Heino, K., Schwartz, D.R., Wolf, G.T., Lacivita, L.P., Ho, L., and Baker, S.R. (1991). In vitro radiation resistance among cell lines established from patients with squamous cell carcinoma of the head and neck. Cancer 67, 2741–2747. Grewal, S.S., Li, L., Orian, A., Eisenman, R.N., and Edgar, B.A. (2005). Mycdependent regulation of ribosomal RNA synthesis during Drosophila development. Nat. Cell Biol. 7, 295–302. Grewal, S.S., Evans, J.R., and Edgar, B.A. (2007). Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway. J. Cell Biol. 179, 1105–1113. Grummt, I., and Pikaard, C.S. (2003). Epigenetic silencing of RNA polymerase I transcription. Nat. Rev. Mol. Cell Biol. 4, 641–649. Guillermo, S., Hong, W., Abdellah, A.-H., Gregory, A.W., Dalia, B.-L., Ludmila, D., Aiping, D., Kong, T.N., David, S., and Yuri, B. (2013). Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5. Biochem. J. 449, 151–159. Hannan, K.M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A., Rothblum, L., Moss, T., Poortinga, G., McArthur, G.A., Pearson, R.B., et al. (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. Hasygar, K., and Hietakangas, V. (2014). p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion. PLoS Genet. 10, e1004764. €inen, T., Seppa € la €, H., Hasygar, K., Auvinen, P., Havula, E., Teesalu, M., Hyo¨tyla , M., Sandmann, T., and Hietakangas, V. (2013). Mondo/ChREBP-MlxOre s ic regulated transcriptional network is essential for dietary sugar tolerance in Drosophila. PLoS Genet. 9, e1003438. Hernandez-Verdun, D. (2006). The nucleolus: a model for the organization of nuclear functions. Histochem. Cell Biol. 126, 135–148. Hietakangas, V., and Cohen, S.M. (2009). Regulation of tissue growth through nutrient sensing. Annu. Rev. Genet. 43, 389–410. Hochstatter, J., Holzel, M., Rohrmoser, M., Schermelleh, L., Leonhardt, H., Keough, R., Gonda, T.J., Imhof, A., Eick, D., Langst, G., et al. (2012). Mybbinding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA. J. Biol. Chem. 287, 24365–24377.

Huang, S. (2002). Building an efficient factory: where is pre-rRNA synthesized in the nucleolus? J. Cell Biol. 157, 739–741. Iadevaia, V., Zhang, Z., Jan, E., and Proud, C.G. (2012). mTOR signaling regulates the processing of pre-rRNA in human cells. Nucleic Acids Res. 40, 2527–2539. Iben, S., Tschochner, H., Bier, M., Hoogstraten, D., Hoza´k, P., Egly, J.-M., and Grummt, I. (2002). TFIIH plays an essential role in RNA polymerase I transcription. Cell 109, 297–306. Johnston, L.A., Prober, D.A., Edgar, B.A., Eisenman, R.N., and Gallant, P. (1999). Drosophila myc regulates cellular growth during development. Cell 98, 779–790. Kantidakis, T., and White, R.J. (2010). A feedback loop between mTOR and tRNA expression? Cell Cycle 9, 2990–2998. Kaser, A., Bogengruber, E., Hallegger, M., Doppler, E., Lepperdinger, G., Jantsch, M., Breitenbach, M., and Kreil, G. (2001). Brix from Xenopus laevis and Brx1p from yeast define a new family of proteins involved in the biogenesis of large ribosomal subunits. Biol. Chem. 382, 1637–1647. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., and Koike, T. (2006). Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol. Cell. Proteomics 5, 749–757. Kos, M., and Tollervey, D. (2010). Yeast pre-rRNA processing and modification occur cotranscriptionally. Mol. Cell 37, 809–820. Koyama, T., and Mirth, C.K. (2016). Growth-blocking peptides as nutritionsensitive signals for insulin secretion and body size regulation. PLoS Biol. 14, e1002392. Laferte´, A., Favry, E., Sentenac, A., Riva, M., Carles, C., and Che´din, S. (2006). The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev. 20, 2030–2040. Larochelle, S., Pandur, J., Fisher, R.P., Salz, H.K., and Suter, B. (1998). Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes Dev. 12, 370–381. Lee, T., Lee, A., and Luo, L. (1999). Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast. Development 126, 4065–4076. €inen, H., and Shore, D. (2009). Growth control and ribosome biogenLempia esis. Curr. Opin. Cell Biol. 21, 855–863. €ngst, G., and Grummt, I. (2006). NoRC-dependent nucleosome posiLi, J., La tioning silences rRNA genes. EMBO J. 25, 5735–5741. Lui, V.W.Y., Hedberg, M.L., Li, H., Vangara, B.S., Pendleton, K., Zeng, Y., Lu, Y., Zhang, Q., Du, Y., Gilbert, B.R., et al. (2013). Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discov. 3, 761–769. Marshall, L., Rideout, E.J., and Grewal, S.S. (2012). Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila. EMBO J. 31, 1916–1930. Marygold, S.J., Roote, J., Reuter, G., Lambertsson, A., Ashburner, M., Millburn, G.H., Harrison, P.M., Yu, Z., Kenmochi, N., and Kaufman, T.C. (2007). The ribosomal protein genes and Minute loci of Drosophila melanogaster. Genome Biol. 8, R216. Mattila, J., Havula, E., Suominen, E., Teesalu, M., Surakka, I., Hynynen, R., €a € na €nen, J., Hovatta, I., Ka €kela €, R., et al. (2015). Mondo-Mlx Kilpinen, H., Va mediates organismal sugar sensing through the Gli-similar transcription factor Sugarbabe. Cell Rep. 13, 350–364. 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. 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. McStay, B., and Grummt, I. (2008). The Epigenetics of rRNA genes: from molecular to chromosome biology. Annu. Rev. Cell Dev. Biol. 24, 131–157. Miyata, Y., Shibata, T., Aoshima, M., Tsubata, T., and Nishida, E. (2014). The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein

Developmental Cell 43, 240–252, October 23, 2017 251

WDR68 and promotes its folding, protein kinase dyrk1a binding, and nuclear accumulation. J. Biol. Chem. 289, 33320–33332. Moir, R.D., and Willis, I.M. (2013). Regulation of pol III transcription by nutrient and stress signaling pathways. Biochim. Biophys. Acta 1829, 361–375. Murano, K., Okuwaki, M., Hisaoka, M., and Nagata, K. (2008). Transcription regulation of the rRNA gene by a multifunctional nucleolar protein, B23/ nucleophosmin, through its histone chaperone activity. Mol. Cell. Biol. 28, 3114–3126. Orlicky, S., Tang, X., Neduva, V., Elowe, N., Brown, E.D., Sicheri, F., and Tyers, M. (2010). An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase. Nat. Biotechnol. 28, 733–737. Orsolic, I., Jurada, D., Pullen, N., Oren, M., Eliopoulos, A.G., and Volarevic, S. (2016). The relationship between the nucleolus and cancer: current evidence and emerging paradigms. Semin. Cancer Biol. 37, 36–50. Peltonen, K., Colis, L., Liu, H., Trivedi, R., Moubarek, M.S., Moore, H.M., Bai, B., Rudek, M.A., Bieberich, C.J., and Laiho, M. (2014). A targeting modality for destruction of RNA polymerase I that possesses anticancer activity. Cancer Cell 25, 77–90. Powers, T., and Walter, P. (1999). Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol. Biol. Cell 10, 987–1000. Rickards, B., Flint, S.J., Cole, M.D., and LeRoy, G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol. Cell. Biol. 27, 937–948. €ki, O., Gre´nman, R., Visakorpi, T., Westermarck, Routila, J., Bilgen, T., Sarama €, S. (2016). Copy number increase of oncoprotein CIP2A is J., and Ventela associated with poor patient survival in human head and neck squamous cell carcinoma. J. Oral Pathol. Med. 45, 329–337. Roux, K.J., Kim, D.I., and Burke, B. (2001). BioID: A Screen for Protein-protein Interactions. Current Protocols in Protein Science (John Wiley). Ruggero, D. (2012). Revisiting the nucleolus: from marker to dynamic integrator of cancer signaling. Sci. Signal. 5, pe38. Sanhueza, G.A.A., Faller, L., George, B., Koffler, J., Misetic, V., Flechtenmacher, C., Dyckhoff, G., Plinkert, P.P., Angel, P., Simon, C., et al. (2012). Opposing function of MYBBP1A in proliferation and migration of head and neck squamous cell carcinoma cells. BMC Cancer 12, 72. Santoro, R., and Grummt, I. (2005). Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation. Mol. Cell. Biol. 25, 2539–2546. Saucedo, L.J., Gao, X., Chiarelli, D.A., Li, L., Pan, D., and Edgar, B.A. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5, 566–571. Schneider, D.A., French, S.L., Osheim, Y.N., Bailey, A.O., Vu, L., Dodd, J., Yates, J.R., Beyer, A.L., and Nomura, M. (2006). RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing. Proc. Natl. Acad. Sci. USA 103, 12707– 12712. Scott, M.S., Troshin, P.V., and Barton, G.J. (2011). NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins. BMC Bioinformatics 12, 317. Shen, J., Jia, W., Yu, Y., Chen, J., Cao, X., Du, Y., Zhang, X., Zhu, S., Chen, W., Xi, J., et al. (2015). Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling. Stem Cells 33, 661–673.

252 Developmental Cell 43, 240–252, October 23, 2017

Suka, N., Nakashima, E., Shinmyozu, K., Hidaka, M., and Jingami, H. (2006). The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner. Nucleic Acids Res. 34, 3555–3567. Tan, B.C.-M., Yang, C.-C., Hsieh, C.-L., Chou, Y.-H., Zhong, C.-Z., Yung, B.Y.-M., and Liu, H. (2012). Epigenetic silencing of ribosomal RNA genes by Mybbp1a. J. Biomed. Sci. 19, 57. Teleman, A.A., Hietakangas, V., Sayadian, A.C., and Cohen, S.M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab. 7, 21–32. Tiefenbo¨ck, S.K., Baltzer, C., Egli, N.A., and Frei, C. (2010). The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling. EMBO J. 29, 171–183. Tollervey, D., Lehtonen, H., Jansen, R., Kern, H., and Hurt, E.C. (1993). Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell 72, 443–457. Tsai, R.Y.L., and Pederson, T. (2014). Connecting the nucleolus to the cell cycle and human disease. FASEB J. 28, 3290–3296. Tschochner, H., and Hurt, E. (2003). Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 13, 255–263. Varjosalo, M., Keskitalo, S., Van Drogen, A., Nurkkala, H., Vichalkovski, A., Aebersold, R., and Gstaiger, M. (2013). The protein interaction landscape of the human CMGC kinase group. Cell Rep. 3, 1306–1320. €, S., Sittig, E., Mannermaa, L., Makela, J.A., Kulmala, J., Loyttyniemi, Ventela E., Strauss, L., Carpen, O., Toppari, J., Grenman, R., et al. (2015). CIP2A is an Oct4 target gene involved in head and neck squamous cell cancer oncogenicity and radioresistance. Oncotarget 6, 144–158. Wang, G.G., Allis, C.D., and Chi, P. (2007). Chromatin remodeling and cancer, part I: covalent histone modifications. Trends Mol. Med. 13, 363–372. Wei, Y., Tsang, C.K., and Zheng, X.F.S. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J. 28, 2220– 2230. Werz, C., Ko¨hler, K., Hafen, E., and Stocker, H. (2009). The Drosophila SH2B family adaptor lnk acts in parallel to chico in the insulin signaling pathway. PLoS Genet. 5, e1000596. Woods, S.J., Hannan, K.M., Pearson, R.B., and Hannan, R.D. (2015). The nucleolus as a fundamental regulator of the p53 response and a new target for cancer therapy. Biochim. Biophys. Acta 1849, 821–829. Yadav, L., Tamene, F., Go¨o¨s, H., van Drogen, A., Katainen, R., Aebersold, R., Gstaiger, M., and Varjosalo, M. (2017). Systematic analysis of human protein phosphatase interactions and dynamics. Cell Syst. 4, 430–444.e5. Yam, A.Y., Xia, Y., Lin, H.-T.J., Burlingame, A., Gerstein, M., and Frydman, J. (2008). Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies. Nat. Struct. Mol. Biol. 15, 1255–1262. Zaragoza, D., Ghavidel, A., Heitman, J., and Schultz, M.C. (1998). Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol. Cell. Biol. 18, 4463– 4470. Zhou, Y., Santoro, R., and Grummt, I. (2002). The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription. EMBO J. 21, 4632–4640.

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies Mouse monoclonal anti-V5

Life Technologies

R960-25

Rabbit polyclonal anti-dPWP1

Casper et al., 2011

N/A

Mouse monoclonal anti-fibrillarin

Abcam

ab4566

Rabbit polyclonal anti-PWP1

Atlas

HPA038707

Rabbit monoclonal anti-PWP1

Abcam

ab190794

Mouse polyclonal anti-POLR1E

Sigma-Aldrich

SAB1400674

Mouse monoclonal anti-H3

Abcam

ab1791

Anti-Histone H4 (acetyl K12)

Abcam

ab46983

Anti-Histone H3 (di methyl K9)

Abcam

ab1220

Rabbit polyclonal anti-HA

Abcam

ab9110

Mouse monoclonal anti-a-Tubulin

Sigma-Aldrich

T8203

Mouse monoclonal anti-dCDK7

Larochelle et al., 1998

19E7

Mouse monoclonal anti-dCDK7

Larochelle et al., 1998

20H5

Rabbit polyclonal anti-GFP

Torrey Pines Biolabs Inc.

TP401

€ et al., 2015 Routila et al., 2016; Ventela

N/A

Biological Samples Tumour samples derived from HNSCC patients Chemicals, Peptides, and Recombinant Proteins Rapamycin

Sigma

37094

Insulin

Sigma

16634

originated from a persistent T4N1M0 Gr 2 cancer on the base of the tongue (Gre´nman et al., 1991)

N/A

Experimental Models: Cell Lines Human: UT-SCC-2 cells (Male)

Human: HeLa cells (Female)

ATCC

ATCC: CCL-2

Human: Flp-In T-REx 293 cells (Female)

ThermoFisher

R78007

Human: U2OS cells (Female)

ATCC

ATCC: HTB-96

D. melanogaster: S2 cells (Male)

ThermoFisher

R69007

D. melanogaster: UAS-Rheb: w[*]; P{w[+mC]=UAS-Rheb.Pa}2

Bloomington Drosophila Stock Center

BDSC: 9688

D. melanogaster: UAS-InR: y[1] w[1118]; P{w[+mC]=UAS-InR.Exel}2

Bloomington Drosophila Stock Center

BDSC, 8262

Experimental Models: Organisms/Strains

D. melanogaster: UAS-Myc

Johnston et al., 1999

N/A

D. melanogaster: UAS-Myc-RNAi

VDRC

2947

D. melanogaster: TOR delP mutant: y[1] w[*]; Tor[DeltaP] P{ry[+t7.2]=neoFRT}40A/CyO

Bloomington Drosophila Stock Center

BDSC: 7014

D. melanogaster: UAS-dPWP1-RNAi

NIG-Fly

6751R-1

D. melanogaster: UAS-dPWP1-RNAi

NIG-Fly

6751R-3

D. melanogaster: nclb1 and nclb2 mutant

Casper et al., 2011

N/A

D. melanogaster: UAS-dPWP1-WT

This paper

N/A

D. melanogaster: UAS-dPWP1-S384A

This paper

N/A

D. melanogaster: UAS-dPWP1-S384E

This paper

N/A

D. melanogaster: mybbp1a mutant

This paper

N/A (Continued on next page)

Developmental Cell 43, 240–252.e1–e5, October 23, 2017 e1

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Cloning primers

This paper, Table S3

N/A

qPCR primers

This paper, Table S3

N/A

dsRNA production primers

This paper, Table S3

N/A

Sequencing primer

This paper, Table S3

N/A

Single-fly PCR primers

This paper, Table S3

N/A

siRNA SMARTpool: siGENOME PWP1

GE Healthcare

M-019744-01-0005

siRNA SMARTpool: siGENOME Nucleolin

GE Healthcare

M-003854-01-0005

siRNA SMARTpool: siGENOME MYBBP1A

GE Healthcare

M-020341-01-0005

Plasmid: GFP-PWP1

This paper

N/A

Plasmid: GFP-PolR1E-V5

This paper

N/A

Plasmid: dPWP1-HA

This paper

N/A

Plasmid: dMybbp1a-V5

This paper

N/A

Plasmid: dPWP1-384A-V5

This paper

N/A

Plasmid: pTO-HA-StrepIII-PWP1-GW-FRT

This paper

N/A

ImageJ

NIH

http://imagej.net

JMP

SAS

https://www.jmp.com/ en_us/software.html

ProgRes CapturePro 2.8.8

Jenoptik

https://www.jenoptik.com/ products/cameras-andimaging-modules/microscopecameras/software-solutions/ image-software-progrescapture-pro

Oligonucleotides

Recombinant DNA

Software and Algorithms

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ville Hietakangas ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Lines Drosophila S2 cells were cultured under 25 C in Shields and Sang M3 medium (Sigma) supplemented with 2% of fetal bovine serum (FBS, LifeTechnologies) and insect media supplement (Sigma) (Havula et al., 2013). UT-SCC-2 cells originated from a persistent T4N1M0 Gr 2 cancer on the base of the tongue (Gre´nman et al., 1991). HeLa, Flp-In T-REx 293 cells, U2OS cells and UT-SCC-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma D-7777) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies), L-glutamate (LifeTechnologies)/GlutaMax (LifeTechnologies), and penicillin/streptomycin (LifeTechnologies). Drosophila Stocks Flies were maintained at 25 C on medium containing malt 6.5% (w/v), semolina 3.2% (w/v), dry baker’s yeast 1.8% (w/v), agar 0.6% (w/v), propionic acid 0.7% (v/v) and Nipagin (methylparaben) 2.4% (v/v) (Havula et al., 2013) or were grown on modified food containing 0.5% (w/v) agar, 2.5% (v/v) Nipagin (methylparaben) in PBS and supplemented with 20% (w/v) dry baker’s yeast or the indicated concentrations of sucrose. Stocks used included UAS-Rheb (BDSC, 9688), UAS-InR (BDSC, 8262), UAS-Myc (Johnston et al., 1999), UAS-Myc-RNAi (VDRC, 2947) TOR delP (BDSC, 7014), tub-Gal4 (Lee et al., 1999), en-Gal4 (Fietz et al., 1995), cg-Gal4 (BDSC, 7011), UAS-dPWP1-RNAi (NIG-Fly, 6751R-1 and 6751R-3), nclb1 and nclb2 (Casper et al., 2011), HsFLP;;FRT82 GFP/ TM3, W;;Actin
METHOD DETAILS Plasmids, RNAi and Cell Culture Drosophila PWP1, MYBBP1A were cloned into modified pMt-V5-HisA (Invitrogen) vectors via ligase independent cloning. In the pMtStrepIII-V5-HisA a C-terminal StrepIII tag was inserted into the vector backbone. In the pMt-StrepIII-HA-HisA, the N-terminal V5 tag was replaced by a 3xHA tag. In both versions, a hygromycin resistance gene was added to the vector backbone. Primers used are listed in Table S3. GFP and V5 tagged human PWP1, POLR1E constructs were generated by the Genome Biology Unit (Biocenter Finland, University of Helsinki). siRNA used were SMARTpool: siGENOME PWP1, Nucleolin and MYBBP1A (GE Healthcare). To analyze phosphorylation, S2 cells were starved for 2 hours with M3 medium, following either inhibition for 2 hours with 1 mM Rapamycin (Sigma, 37094) followed by stimulation for 10 minutes with 10 mg/ml insulin (Sigma, 16634) or insulin stimulation alone. To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days. The transfections were performed using Lipofectamine 2000, Lipofectamine RNAiMAX (LifeTechnologies) or Effectene (Qiagen) according to the manufacturer’s protocol. Drosophila Genetics To generate mybbp1a flies, pL100 construct expressing sgRNA was purchased from GEPG at Harvard Medical School and injected into embryos of Bloomington Stock 51324 carrying vas-Cas9 and GFP. After injection, each male founder was crossed to FM7 virgin flies separately. 3 GFP negative F1 female virgins from each founder carrying FM7 were individually crossed to FM7, GFP to establish GFP balanced stocks. Genomic DNA was then extracted from single flies of lethal stocks followed by PCR amplification and molecular verification using T7 endonuclease I. To characterize positive candidates from T7 endonuclease I assay, Sanger sequencing was performed on PCR products amplified from genomic DNA extracted from GFP negative larvae using the same PCR primers. The UAS-dPWP1-WT and UAS-dPWP1-S384A transgenic flies were made by subcloning the cDNA into the pUAST transformation vector, and injected to 2nd chromosome landing site 25C6 by Fly Facility, Department of Genetics, University of Cambridge. To generate mosaic clones, larvae (genotype: HsFLP/+;;UAS-dPWP1-RNAi/Actin
samples were washed and mounted with Vectashield Mounting Medium with DAPI (Mediq), and imaged using a Zeiss LSM 700 microscope. Alanine Scanning The mutation of all dPWP1 serines and threonines into alanines was done with GeneArt Subcloning & Express Cloning Service (Life Technologies). Co-Immunoprecipitations and Immunoblotting For the co-immunoprecipitations, cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.5% NP-40) with Pierce Protease Inhibitor Tablets (Thermo) alone or with Pierce Protease Inhibitor (Thermo). Anti-V5 beads were made from antiV5 antibody and 50% protein A/G sepharose beads (Amersham). Lysate was incubated 2 hours with anti-V5 beads or anti-HA Agarose Conjugate (Sigma, A2095). After washing the beads 5 times with NP-40 lysis buffer, 2X Laemmli buffer was added and the protein was denatured by boiling for 3 minutes. Pulldown and lysate samples were resolved on SDS-PAGE, and analysed by Western blotting. Chromatin Immunoprecipitation HeLa or U2OS cells were washed with PBS and fixed with 1% formaldehyde for 10 minutes (RT). The fixation was quenched by adding glycine to 125 mM. The cells were washed twice with PBS before collecting and were frozen at -80  C for at least one hour. After thawing, cells were lysed on ice for 15 minutes in nuclear lysis buffer (EDTA 10 mM, Tris-HCl 50 mM pH=8.1, 0.5% Empigen B, SDS 1%), sonicated with NGS bioruptor sonicator (resulting DNA fragments ranging 500-800bp). After 10X dilution in IP buffer (EDTA 2 mM, NaCl 150 mM, Tris-HCl 20 mM pH=8.1, Triton-X100 0.1%, with protease inhibitors), pre-cleaning by 3 hour incubation with 50% protein A-Sepharose CL-4B bead (Fisher Scientific) slurry (+4 C), the lysate was coupled with the antibody over-night (+4  C). The antibody-coupled lysates were incubated with blocked (incubated over-night at +4  C with IP buffer supplemented with 1 mg/ml BSA and 5 mg salmon sperm ssDNA) 50 % protein A-Sepharose CL-4B bead slurry for 3 hour (+4  C), and washed once with three buffers containing protease inhibitors (Buffer I: EDTA 2 mM, Tris-HCl 20 mM pH=8.1, SDS 0.1 %, Triton X-100 1 %, NaCl 150 mM. Buffer II: EDTA 2 mM, Tris-HCl 20 mM pH=8.1, SDS 0.1 %, Triton X-100 1 %, NaCl 500 mM. Buffer III: EDTA 1 mM, Tris-HCl 10 mM pH=8.1, LiCl 250 mM, Deoxycholate 1 %, NP-40 1 %), followed by two washes with 1xTE. The chromatin was released by incubating the beads for 15 minutes with 75 ml of extraction buffer (SDS 1 %, NaHCO3 0.1 M) and vortexing (RT). The beads were centrifuged at 450 g (RT), and the supernatant was collected. Another 75 ml of extraction buffer was added and the supernatant was collected again. The combined supernatant samples and inputs were incubated with 50 mg/ml RNaseA for 1 h (37  C), and then over-night (65  C). After treated with 0.5 mg/ml proteinase K for 2 h (45  C), the DNA was purified using the MinElute kit (Qiagen) and was subjected to qRT-PCR. (Mattila et al., 2015). Single-Step Affinity Purification The PWP1 affinity purifications were performed as described previously (Varjosalo et al., 2013). HEK293 Flp-In TREx cells expressing N-terminal strep-tagged (pTO-HA-StrepIII-GW-FRT) PWP1 were induced with 2 mg/ml of tetracycline for 24 hrs. After that cells were pelleted, snap frozen in liquid nitrogen and stored at -80 C. For affinity purification, cell pellets were suspended into 3 ml of HENN buffer (50 mM HEPES pH 8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% NP40) with 1.5 mM Na3VO4, 1 mM PMSF, 1x protease inhibitors cocktail (Sigma). Cells were incubated on ice for 15 minutes. Lysates were centrifuged and cleared supernatant was loaded onto Spin-columns (Bio-Rad, USA) packed with 200 ml of Strep-Tactin beads (IBA GmbH). The beads were washed 3x with 1 ml of HENN buffer with 1.5 mM Na3VO4, 1 mM PMSF, 1x protease inhibitors cocktail (Sigma) and 4x with 1 ml of HENN buffer. The bound proteins were eluted from beads with 900 ml of HENN buffer supplemented with 0.5 mM biotin. For HA-purification of dPWP1, lysates were incubated with 100 ml of Anti-HA Agarose beads (Sigma-Aldrich) for 2 hrs at 4 C on a rotary shaker. Beads are placed in Bio-Rad Spin-columns and washed similarly as described above. The proteins are eluted batch wise with 500 ml of 0.2 M Glycine (pH 2.5). BioID The proximity-dependent biotin identification (BioID) of the PWP1 interactors was performed as described in (Yadav et al., 2017). BioID-tagged (pTO-BirA-myc-GW-FRT) PWP1 was induced with 2 mg/ml of tetracycline and cells were treated with 50 mM biotin for 24 hrs. After that cells were pelleted, snap frozen in liquid nitrogen and stored at -80 C. For affinity purification, cell pellets were suspended into 3 ml of HENN-lysis buffer (50 mM HEPES pH 8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% NP40) supplemented with 0.1% SDS and 80 U/ml Benzonase Nuclease (Santa Cruz Biotechnology, Dallas, TX). Cells were incubated on ice for 15 minutes and additionally sonicated on ice (3 cycles of 3 minute each). Lysates were centrifuged and cleared supernatant was loaded onto Spin-columns (Bio-Rad, USA) packed with 200 ml of Strep-Tactin beads (IBA GmbH). The beads were washed 3x with 1 ml of HENN buffer with 1.5 mM Na3VO4, 1 mM PMSF, 1x protease inhibitors cocktail (Sigma) and 4x with 1 ml of HENN buffer. The bound proteins were eluted from beads with 900 ml of HENN buffer supplemented with 0.5 mM biotin. Mass Spectrometry Samples for liquid chromatography MS were prepared as follows: TCEP [Tris(2-carboxyethyl)phosphine hydrochloride was added to the eluates to a final concentration of 5 mM, and the samples were incubated for 30 min at 37 C. To block cysteine residues, e4 Developmental Cell 43, 240–252.e1–e5, October 23, 2017

iodoacetamide was added to a final concentration of 10 mM and the samples were incubated at room temperature in the dark for 30 min. A total of 1 mg Sequencing Grade Modified Trypsin (Promega) was added and the samples were incubated overnight at 37 C. Tryptic digests were quenched with 10% trifluoroacetic acid (TFA), concentrated and purified by reverse-phase chromatography MicroSpin Column (C18, Nest Group) and eluted with 50% acetonitrile, 0.1% TFA. The eluted samples were dried in a vacuum centrifuge and reconstituted to a final volume of 30 ml with 0.1% TFA, 1% acetonitrile and vortexed thoroughly. Mass spectrometry analysis was performed on an Orbitrap Elite ETD mass spectrometer (Thermo Scientific) using the Xcalibur version 2.7.1 coupled to a Thermo Scientific nLCII nanoflow system (Thermo Scientific) via a nanoelectrospray ion source. Solvents for liquid chromatography MS separation of the digested samples were as follows: solvent A consisted of 0.1% formic acid in water (98%) and acetonitrile (2%), and solvent B consisted of 0.1% formic acid in acetonitrile (98%) and water (2%). From a thermostatted microautosampler, 8 ml of the tryptic peptide mixture (corresponding to 20% of the final eluate) was automatically loaded onto a 15 cm fused silica analytical column with an inner diameter of 75 mm packed with C18 reversed-phase material (Thermo Scientific), and the peptides were eluted from the analytical column with a 40 min gradient ranging from 5% to 35% solvent B, followed by a 10 min gradient from 35% to 80% solvent B at a constant flow rate of 300 nl/min. The analyses were performed in a data-dependent acquisition mode using a top 10 collision-induced dissociation (CID) method. Dynamic exclusion for selected ions was 30 s. No lock masses were employed. Maximal ion accumulation time allowed on the Orbitrap Elite in CID mode was 200 ms for MSn in the Ion Trap (IT) and 200 ms in the Fourier transform mass spectrometer (FTMS). Automatic gain control was used to prevent overfilling of the ion trap and was set to 10,000 in MSn mode for the Ion Trap and 106 ions for a full FTMS scan. Intact peptides were detected in the Orbitrap at 60,000 resolution. Peak extraction and subsequent protein identification was achieved using Proteome Discoverer software (Thermo Scientific). Calibrated peak files were searched against the human component of UniProtKB/SwissProt database (http://www.uniprot.org) by a SEQUEST search engine at default FDR settings (<0.05). Error tolerances on the precursor and fragment ions were ±15 ppm and ±0.6 Da, respectively. Database searches were limited to fully tryptic peptides with maximum one missed cleavage. Carbamidomethyl (+57.021464 Da) cysteine was set as static modification whereas oxidation (+15.994491 Da) of methionine and biotinylation (+226.078 Da) of lysine or n-terminus were set as variable modifications. 4sU-Labeling of RNA Cells were incubated with 500 mM of 4sU for 30 minutes, and total RNA was extracted using a Nucleospin RNA II kit (MachereyNagel), biotinylated with 0.2mg/ml EZ-Link-Biotin-HPDP (Thermo) and purified as previously described (Do¨lken et al., 2008). RNA samples were resolved on 1% agarose/Formaldehyde gel, visualized with Midori Green (NIPPON Genetics) or ethidium bromide staining, then transferred to Hybond XL membrane (GE) and the biotin-bound horseradish peroxidase was visualized by Amersham ECL Prime Western Blotting Detection Reagent (GE). Immunohistochemistry Formalin-fixed, paraffin-embedded sections of human HNSCC tumor specimens were cut into 6 mm thin sections, deparaffinised and thereafter rehydrated. Epitope retrieval was then proceeded in 10 mM Tris-EDTA-buffer (pH 9) during 4 min in microwave oven 4 min 850 W followed by 15 min at a lower power (150 W). After blocking with 3% BSA PBS for 10 min the slides were rinsed in Tris-HCl (pH 7.4), and incubated overnight with primary antibody against PWP1 (1:100 Life Technologies). Control slides were incubated with normal nonimmunized appropriate animal serum. The samples were then incubated appropriate secondary antibody (Dako EnVision anti-rabbit or anti-mouse) for 30 min and 10 min in DAB+ liquid Dako (K3468). QUANTIFICATION AND STATISTICAL ANALYSIS JMP was used for the ANOVA analysis of Figure 6I, and for all other experiments Student’s t-test were performed for statistical calculations. n indicates the number of biological replicates and is detailed in the figure legends. Error bars indicate the standard deviations. Fluorescence and compartment size was quantified by ImageJ. To calculate the pupal volume, the following formula was used: 4/3p(L/2)(W/2)2 (L, length; W, width). The length and width were measured with the ProgRes CapturePro 2.8.8 program (Jenoptik).

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