SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport

Article

SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport Highlights d

Starvation causes redistribution of eukaryotic polyadenylated transcripts

d

SIRT1 is a negative regulator of poly(A)RNA transport

d

AMPK triggers starvation-induced poly(A)RNA nuclear retention

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Shutting down poly(A)RNA export offers eukaryotes an energy-saving shortcut

Authors Peipei Shan, Guangjian Fan, Lianhui Sun, ..., Yongping Cui, Shengping Zhang, Chuangui Wang

Correspondence [email protected] (S.Z.), [email protected] (C.W.)

In Brief The addition of poly(A)-tail to mRNA is a posttranscriptional modification process required for most eukaryotic mRNA export and translation. Shan et al. show that energy starvation results in marked inhibition of poly(A)RNA export, which offers eukaryotes a shortcut to tailor protein synthesis and energy consumption to adapt to energy stress.

Shan et al., 2017, Current Biology 27, 1–14 August 7, 2017 ª 2017 Elsevier Ltd. http://dx.doi.org/10.1016/j.cub.2017.06.040

Please cite this article in press as: Shan et al., SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport, Current Biology (2017), http://dx.doi.org/10.1016/j.cub.2017.06.040

Current Biology

Article SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport Peipei Shan,1,9 Guangjian Fan,1,9 Lianhui Sun,1,9 Jinqin Liu,2 Weifang Wang,2 Chen Hu,3 Xiaohong Zhang,3 Qiwei Zhai,4 Xiaoyu Song,5 Liu Cao,5 Yongping Cui,6 Shengping Zhang,1,* and Chuangui Wang1,7,8,10,* 1Institute

of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, East China Normal University, Shanghai 200241, China 3Department of Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, 4100 John R, Detroit, MI 48201, USA 4Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 5Key Laboratory of Medical Cell Biology, College of Translational Medicine, China Medical University, Shengyang 110000, China 6Key Laboratory of Cellular Physiology Ministry of Education, Shanxi Medical University, Shanxi 030001, China 7Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China 8Shanghai Key Laboratory of Fundus Diseases, 100 Haining Road, Shanghai 200080, China 9These authors contributed equally 10Lead Contact *Correspondence: [email protected] (S.Z.), [email protected] (C.W.) http://dx.doi.org/10.1016/j.cub.2017.06.040 2Shanghai

SUMMARY

Most eukaryotic mRNAs are polyadenylated in the nucleus, and the poly(A)-tail is required for efficient mRNA export and translation. However, mechanisms governing mRNA transport remain unclear. Here, we report that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 acts as an energy sensor and negatively regulates poly(A)RNA transport via deacetylating a poly(A)-binding protein, PABP1. Upon energy starvation, SIRT1 interacts with and deacetylates PABP1 and deactivates its poly(A) RNA binding, leading to nuclear accumulation of PABP1 and poly(A)RNA and thus facilitating eukaryotic cells to attenuate protein synthesis and energy consumption to adapt to energy stress. Moreover, AMPK-directed SIRT1 phosphorylation is required for energy starvation-induced PABP1-SIRT1 association, PABP1 deacetylation, and poly(A)RNA nuclear retention. In addition, the SIRT1-PABP1 association is not specific to energy starvation but represents a common stress response. These observations provide insights into dynamic modulation of eukaryotic mRNA transport and translation, suggesting that the poly(A)-tail also provides a basis for eukaryotes to effectively shut down mature mRNA transport and thereby tailor protein synthesis to maintain energy homeostasis under stress conditions. INTRODUCTION In eukaryotic cells, transcription occurs in the nucleus, whereas translation occurs in cytoplasm. The addition of poly(A)-tail to mRNA is a common posttranscriptional modifi-

cation process required for mRNA export and translation [1, 2]. The poly(A)-binding proteins (PABPs) play important roles in mRNA polyadenylation, translation, and nuclear export [3–7]. There are seven PABPs in humans, including PABPC1, PABPC3, PABPC4, PABPC4L, PABPC5, ePABP, and PABPN1 [6]. PABPC1 (hereafter referred to as PABP1) has four N terminus RNA recognition motifs (RRMs), a linker region, and a C-terminal PABC domain. The RRMs are necessary for interaction with poly(A) [8, 9]. PABP1 RRMs also bind to some proteins involved in mRNA translation and metabolism, such as Paip1, Paip2, GW182, and eIF4G [6, 7]. The C-terminal PABC domain is required for recruitment of translation factors to the mRNA poly(A) tail [6, 7, 10]. PABP1 is a nucleocytoplasmic shuttle protein with a predominantly cytoplasmic localization under normal growth conditions [5, 11]. In cytoplasm, by simultaneous binding to poly(A) and eIF4G, PABP1 brings a ‘‘closed loop’’ of mRNA conformation [3, 12], which facilitates 40S ribosomal subunit recruitment, stabilizes mRNA, and promotes translation [13–16], whereas under stress conditions, such as heat shock, UV irradiation [17, 18], or viral infection [19, 20], PABP1 accumulates mainly in the nucleus. The functional consequences of nuclear accumulation of PABP1 are mRNA hyperadenylation and inhibition of poly(A)RNA export, which lead to restriction of protein expression [21, 22]. Therefore, based on its subcellular localization, PABP1 may play opposite roles in regulating gene expression. Silent information regulator (Sir2)-like family deacetylases (also known as sirtuins) play important roles in the regulation of cellular stress resistance, genomic stability, and energy metabolism [23–26]. In mammals, there are seven Sirtuins (SIRT1–7). SIRT1 regulates energy metabolism and stress response by deacetylating histone and a large number of transcription factors, such as p53, E2F1, and FOXOs [25–30]. The relevance of SIRT1 to metabolic status and stress response greatly depends on its effect on transcription [31]. Interestingly, loss of SIRT1 results in increased phosphorylation of translation initiation factor eIF2a [32], and the sirtuin activator resveratrol Current Biology 27, 1–14, August 7, 2017 ª 2017 Elsevier Ltd. 1

Please cite this article in press as: Shan et al., SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport, Current Biology (2017), http://dx.doi.org/10.1016/j.cub.2017.06.040

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induces AMPK activation and thus contributes to the inhibition of 4E-BP1 signaling [33], suggesting a possible role of SIRT1 in regulating translation. Results described below show that energy starvation causes poly(A)RNA nuclear retention in eukaryotes. Molecularly, upon starvation, SIRT1 deacetylates PABP1 and impairs its poly(A) RNA binding, which causes poly(A)RNA nuclear export and translation inhibition, leading to energy saving. We suggest that shutting down poly(A)RNA export offers eukaryotes a shortcut to tailor protein synthesis and energy consumption to adapt to energy stress. RESULTS Energy Starvation Induces Poly(A)RNA Nuclear Accumulation Protein synthesis is a highly energy-consuming process. Because export of mature mRNA from the nucleus is a critical regulatory step in gene expression, we propose that switching off mRNA nuclear export may provide eukaryotes a shortcut to tailor protein synthesis to preserve energy homeostasis under starvation. The poly(A) tail is found at the 30 end of most fully processed eukaryotic mRNAs; we therefore checked poly(A)RNA subcellular distribution using a Cy3-labeled oligo(dT) probe. Whereas poly(A)RNA is mainly distributed in the cytoplasm at normal condition, strikingly, we observed that most of the poly(A)RNAs were accumulated in the nucleus under glucose deprivation (GD) conditions (Figure 1A). Similar result was observed in cells treated with 2-deoxy-D-glucose (2DG), a glucose metabolism inhibitor that mimics the effect of energy starvation (Figure 1B). Of note, a-amanitin (an inhibitor of RNA polymerase II) treatment marked blocked GD-induced nuclear accumulation of poly(A)RNA (Figure S1A). In addition, we observed no cell size alteration under GD treatment (12 hr; Figure S1B). Together, these results demonstrate that poly(A)RNA nuclear export can be blocked by energy starvation. SIRT1 Regulates Starvation-Induced Poly(A)RNA Redistribution SIRT1 functions as a nutrient-sensing deacetylase [34], and its expression is augmented following fasting [35]. Interestingly, we observed that GD-induced poly(A)RNA nuclear accumulation is markedly inhibited in SIRT1-knockout (KO) or -knockdown cells (Figures 1C and 1D). Evaluation of the average nuclear/ cytoplasmic (N/C) ratio of poly(A)RNA indicates that SIRT1 KO

led to a significant inhibition of GD-induced poly(A)RNA nuclear accumulation (Figure 1C, right panel). Overexpression of wildtype (WT) SIRT1, but not a deacetylase-defective mutant of SIRT1 (SIRT1-363A), led to marked poly(A)RNA nuclear accumulation (Figure 1E). Pharmacological activation of SIRT1 using resveratrol resulted in marked poly(A)RNA nuclear accumulation (Figure 1F). These results indicate that SIRT1 is required for GD-induced poly(A)RNA nuclear accumulation. Given that brain is the most energy-consuming organ in animals, we next examined poly(A)RNA distribution in the cells of mouse cerebral cortex during fasting using biotinylated oligo(dT) probe. Results show that a short-term fasting (24 hr) induced a marked nuclear accumulation of poly(A)RNA in cerebral cortex of WT, but not SIRT1-KO mice, and re-feeding led to cytoplasmic redistribution of poly(A)RNA (Figure 1G), suggesting that the subcellular poly(A)RNA distribution in brain tissue is dynamically regulated in response to energy variation, and SIRT1 is required for this response. Of note, in the low-energy-consumption tissues, such as lung and colon [36], 24-hr fasting led to a slight increase in the N/C ratio of poly(A)RNA in the WT, but not SIRT1KO, mice (Figure S2), suggesting that poly(A)RNA subcellular distribution is regulated by the energy status of tissues. SIRT1 Interacts with PABP1 and Regulates Its Distribution and Activity in Translation A previous study indicated that a slight elevation in the levels of nuclear PABP1 is sufficient to cause poly(A)RNA nuclear accumulation [21]. We therefore tested whether SIRT1 regulates poly(A)RNA subcellular distribution via PABP1. Coimmunoprecipitation experiments from transiently transfected 293T cells show that SIRT1 coprecipitated with PABP1, but not PABPN1 (Figure 2A). The endogenous SIRT1-PABP1 association is also detectable, and the binding is not interfered by RNase A (a pyrimidine-specific RNase; Figure 2B) or RNase I (a RNase degrades all RNA dinucleotide bonds) treatment (data not shown), suggesting that the SIRT1-PABP1 interaction is not RNA mediated. The glutathione S-transferase (GST)-pull-down assay confirmed the direct SIRT1-PABP1 interaction in vitro (Figure 2C). A semi-in-vivo pull-down assay using the E.-coli-expressed GST-SIRT1 protein and the lysates of Flag-PABP1 deletion mutant overexpressing cells confirmed that second RRM motif of PABP1 is required for binding to SIRT1 (Figure 2D). Furthermore, the SIRT1-363A mutant dramatically attenuates its association with PABP1 (Figure 2E). These results indicate that PABP1 is a SIRT1-interacting protein.

Figure 1. SIRT1 Regulates Poly(A)RNA Redistribution under Energy Starvation (A and B) HeLa cells treated with glucose deprivation (GD) (A) or 2-DG (12.5 mM; B) for the indicated times were stained for poly(A)RNA (red) using Cy3-oligo(dT)40 and viewed by fluorescence microscopy (scale bar 50 mm). Nuclei were stained with DAPI. (C and D) Images show poly(A)RNA staining in wild-type (WT) and SIRT1 knockout (KO) mouse embryonic fibroblast (MEF) cells at passages 3 or 4 (C) or HeLa cells with stable knockdown of SIRT1 (ShR) or vector control (ShN) (D), in the presence (untreated) or absence (GD) of glucose for 6–12 hr (scale bar 50 mm). Data of nuclear/cytoplasmic (N/C) ratio of poly(A)RNA staining show the mean ± SD for 40–60 cells from three independent experiments. SIRT1 expression was detected by western blot. (E) HeLa cells transfected with vector, Flag-SIRT1, and Flag-SIRT1-363A plasmids were stained for poly(A)RNA (red), Flag-SIRT1(green), and nuclei (blue) and viewed by fluorescence microscopy (scale bar 20 mm). Arrowheads indicate Flag-SIRT1, but not Flag-SIRT1-363A, overexpression induces poly(A)RNA nuclear accumulation. (F) Images show poly(A)RNA staining in HeLa cells treated with or without resveratrol (100 mM; 12 hr). The scale bar represents 20 mm. (G) Immunohistochemical staining of poly(A)RNA in the cerebral cortex of WT and SIRT1-KO mice under normal feeding, fasting (24 hr), or 1-day food re-feeding after 24 hr fasting (scale bar 50 mm). The boxed enlargements show poly(A)RNA localization. Tissues hybridized without probe were used as negative control. See also Figures S1 and S2.

Current Biology 27, 1–14, August 7, 2017 3

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We next tested whether SIRT1 regulates PABP1 subcellular distribution. Strikingly, overexpression of WT SIRT1, but not SIRT1-363A mutant, induced a marked nuclear accumulation of endogenous PABP1 (Figure 2F). In contrast, SIRT1 overexpression showed no effect on cytoplasmic distribution of PABP1-associated proteins, including eIF4G and eIF4B (Figures S3A and S3B), suggesting that SIRT1 overexpression causes a spatial separation between PABP1 and translation initiation factors. Moreover, resveratrol treatment led to marked PABP1 nuclear accumulation (Figure 2G). Therefore, overexpression or activation of SIRT1 induces nuclear accumulation of PABP1. The function of PABP1 in translation activation depends mostly on its interacting with poly(A)mRNA and the eIF4F translation initiation complex [8, 37]. We next tested whether SIRT1 negatively regulates PABP1 activity in translation. Results show that overexpression of SIRT1, but not SIRT1-363A mutant, markedly repressed PABP1-poly(A)RNA binding (Figure 2H). Moreover, overexpression of WT SIRT1 (but not SIRT1-363A mutant) resulted in a marked downregulation of PABP1-eIF4G complex formation (Figure 2I), whereas SIRT1 overexpression had no effect on eIF4G-eIF4B binding (Figure S3C). Furthermore, resveratrol treatment caused marked decrease in 35S-methionine incorporation into nascent proteins (Figure 2J) and PABP1-poly(A)RNA binding (Figure 2K). Together, these results indicate that forced expression or activation of SIRT1 induce PABP1 nuclear accumulation and thereby inhibit its activity in poly(A)mRNA binding and translation. SIRT1 Regulates PABP1 Distribution via Deacetylation A previous study identified that PABP1 is post-translationally modified by arginine methylation [38]. A mass spectrometry study has shown that human PABP1 protein contains four acetylated lysine residues (K95, K188, K312, and K606) [39]. Next, we examined whether SIRT1 regulates PABP1 function via deacety-

lation. Results show that overexpression of WT SIRT1 (but not SIRT1-363A mutant) markedly decreased PABP1 acetylation (Figure 3A), SIRT1-knockdown led to an increased PABP1 acetylation (Figure 3B), and WT SIRT1 (but not SIRT1-363A mutant) deacetylated PABP1 in vitro (Figure 3C), indicating that PABP1 is a substrate of SIRT1. Moreover, site-directed mutagenesis (K-to-R) analysis shows that K95 is a major acetylation site of PABP1 (Figure 3D). Next, we generated a polyclonal antibody that recognizes K95-acetylated PABP1 (K95AC). Results show this antibody recognizes the WT Flag-PABP1 but neither the acetylation-deficient K95R nor the acetylation-mimicking K95Q mutants that ectopically expressed in 293T cells (Figure 3E) nor the recombinant E.-coli-expressed His-tagged PABP1 (Figure 3F), demonstrating that this antibody specifically recognizes K95-acetylated PABP1. Moreover, PABP1-K95AC was markedly decreased in cells overexpressing WT SIRT1 (but not SIRT1363A mutant; Figure 3G), and treatment of the Sirtuin inhibitor nicotinamide (NAM) led to upregulation of PABP1-K95AC (Figure 3H). These results demonstrate that SIRT1 deacetylates PABP1 at K95. Next, we tested whether PABP1 deacetylation regulates its subcellular distribution. Results show that the PABP1 K95R mutant was localized mainly in the nucleus, whereas other three site mutants (K188R, K312R, and K606R) were mainly cytoplasmic (Figure 3I). Moreover, coexpression of SIRT1 induced a marked nuclear accumulation of the WT PABP1, but not the K95Q mutant (Figure 3J). Collectively, the above results suggest that SIRT1 deacetylates PABP1 at K95 and thereby regulates its subcellular distribution. PABP1-K95 Deacetylation Leads to Suppression of Poly(A)RNA Export and Translation To examine the role of PABP1 deacetylation, we generated stable PABP1-knockdown and Flag-PABP1 (including WT, K95R,

Figure 2. SIRT1 Interacts with PABP1 and Regulates Its Distribution and Activity in Translation (A) 293T cells were cotransfected with indicated plasmids for 24 hr; cell lysates were immunoprecipitated using FLAG-M2 beads followed by western blot analysis. (B) HeLa cells were lysed in the absence or in presence of RNase A (10 mg/mL) as indicated. Cell extracts were precipitated using anti-PABP1 antibody or with immunoglobulin G (IgG) (Mock IP) followed by western blotting analysis. (C) SIRT1 interacts with PABP1 in vitro. Recombinant E.-coli-expressed His-tagged PABP1 protein was incubated with GST-SIRT1 or GST proteins in the presence of RNase A (10 mg/mL) at 4 C for 4 hr followed by GST pull-down and western blot. (D) PABP1-SIRT1 interaction domains in PABP1 were determined by incubating recombinant E.-coli-expressed GST-SIRT1 protein with lysates of 293T cells overexpressing Flag-PABP1 deletion mutants, followed by immunoprecipitation with FLAG-M2 beads and western blot. (E) 293T cells cotransfected with indicated plasmids were lysed and immunoprecipitated with FLAG-M2 beads followed by western blot with anti-SIRT1 and antiFlag antibodies. (F) HeLa cells were transfected with vector, Flag-SIRT1, or Flag-SIRT1-363A for 20 hr; fixed; and stained for anti-Flag (red), anti-PABP1 (green), and DAPI (blue) and visualized by microscopy (scale bar 20 mm). (G) HeLa cells treated with resveratrol (100 mM; 0–12 hr) were immunostained using anti-PABP1 (green) antibody and visualized by microscopy (scale bar 20 mm). (H) 293T cells were cotransfected with indicated plasmids for 24 hr. Poly(A)RNA was precipitated using poly(U)-conjugated agarose beads in the presence or absence of RNase A (10 mg/mL). The amount of PABP1 associated with poly(A)RNA was assessed by western blot analysis and quantified by densitometry of the immunoblots. The graph shows the relative ratio of coprecipitated PABP1 versus the amount of the precipitated poly(A)RNA (PABP1/RNA ratio) in each sample (n = 3; **p < 0.01). (I) 293T cells cotransfected with indicated plasmids were lysed and immunoprecipitated with FLAG-M2 beads. Coprecipitated GFP-tagged eIF4Gs were blotted for GFP. (J) HeLa cells were incubated with the indicated doses of resveratrol for 12 hr before the addition of 35S-methionine-protein labeling mix (20 mCi/mL; 1 hr). Cells were harvested after labeling, and the radioactivity incorporation of newly synthesized proteins were analyzed by SDS-PAGE and autoradiography (upper panel). Actin (input control) was detected by western blot (lower panel). (K) HeLa cells treated with or without resveratrol (100 mM; 12 hr) were lysed and precipitated with poly(U)-conjugated agarose beads followed by western blot using anti-PABP1 antibody. Coprecipitated endogenous PABP1was blotted with anti-PABP1 antibody. The relative PABP1/RNA ratio was analyzed as in (H). See also Figure S3.

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Please cite this article in press as: Shan et al., SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport, Current Biology (2017), http://dx.doi.org/10.1016/j.cub.2017.06.040

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and K95Q)-rescued cell lines. We observed that PABP1 knockdown led to marked poly(A)RNA nuclear accumulation, and recomplementation of either PABP1-WT or -K95Q (but not PABP-K95R) caused the redistribution of poly(A)RNA from the nucleus to the cytoplasm (Figure 4A), suggesting that PABP1 is indispensable in poly(A)RNA nuclear-cytoplasmic transportation and PABP1 K95AC is required for the activity of PABP1 in poly(A) RNA nuclear export. Moreover, PABP1 knockdown caused significant growth retardation (Figure 4B), and recomplementation of PABP1-WT, but not PABP1-K95R, significantly rescued the cell growth retardation induced by PABP1 knockdown (Figure 4C). Furthermore, PABP1-K95R mutant shows declined poly(A)RNA binding (Figure 4D), and the PABP1-K95R, but not -K95Q mutant, exhibited a reduced chance to form a complex with translation initiation factor eIF4G (Figure 4E). In addition, 35S-methionine-protein labeling of newly synthesized proteins revealed that both the PABP1-knockdown and the PABP-K95R-recomplemented cells exhibited lower levels of 35 S-methionine incorporation in comparing with the normal and PABP1-WT recomplemented cells (Figure 4F). Together, these results suggest that PABP1 K95 deacetylation decreases its ability in poly(A)mRNA binding and export and leads to a spatial separation of the translation initiation complex from poly(A) mRNA and PABP1, thereby resulting in the repression of protein synthesis and cell growth.

in the PABP1 K95Q mutant-rescued, cell lines, and the PABP1-K95Q mutant retained in the cytoplasm under starvation (Figure 5D). Combining these observations with the facts that PABP1-K95 deacetylation leads to nuclear retention of poly(A) RNA, we conclude that SIRT1 limits mRNA transport via deacetylating PABP1 under GD. Consistently, immunohistochemistry staining of PABP1 and PABP1-K95AC in the cerebral cortex sections of WT and SIRT1-KO mice revealed that both PABP1 and PABP1-K95AC exhibited obvious cytoplasmic distribution under normal feeding conditions, whereas a 24-hr fasting caused marked nuclear accumulation of PABP1 coupled with decreased PABP1-K95AC in normal, but not SIRT1-KO, mice (Figure 5E). Next, we tested whether SIRT1-directed PABP1 deacetylation tricks cells into an energy-saving mode. Results show that PABP1 knockdown led to a significant downregulation of intracellular ATP consumption, and reconstitution of PABP1-K95Q, but not PABP1-WT or -K95R, led to a rapid decrease of intracellular ATP level under GD conditions (Figure 5F), suggesting that SIRT1-induced PABP1 K95 deacetylation under starvation contributes to energy saving. In addition, we observed that a short period of glucose re-feeding after GD led to marked PABP1SIRT1 dissociation coupled with the nuclear export of both poly(A)RNA and PABP1 (Figure S4), indicating that the SIRT1PABP1 binding is dynamically regulated to fine-tune mRNA export to adapt to the variation of the energy supply.

Starvation Induces SIRT1-Dependent PABP1 Deacetylation and Nuclear Accumulation Next, we examined whether the endogenous SIRT1 mediates poly(A)RNA transport via deacetylating PABP1 upon starvation. Results show that GD caused a marked nuclear accumulation of endogenous PABP1 (Figure 5A) coupled with increased PABP1-SIRT1 association (Figure 5B), and PABP1-K95AC decreased under GD treatment in a SIRT1-dependent manner (Figure 5C). Moreover, GD treatment caused marked poly(A) RNA nuclear accumulation in the PABP1WT-rescued, but not

AMPK Triggers PABP1 and Poly(A)RNA Redistribution via Phosphorylating SIRT1 Our previous study revealed that GD induces phosphorylation of SIRT1 by AMPK [40]. In this study, we observed that AICAR (AMPK activator) treatment led to marked nuclear accumulation of both PABP1 and poly(A)RNA (Figures 6A and 6B). GD treatment led to enhanced SIRT1 phosphorylation at T530 and T719, but not at S14 (Figure 6C). The phosphorylation-defective SIRT1-T530A, but not -T719A, mutant markedly attenuated GDinduced increment of PABP1-SIRT1 association (Figure 6D).

Figure 3. SIRT1 Regulates PABP1 Transport via Deacetylation (A) 293T cells cotransfected with indicated plasmids were lysed and immunoprecipitated with FLAG-M2 beads followed by western blot analysis. PABP1 acetylation (PABP1AC) was detected using anti-acetyl-lysine or anti-Flag antibodies. (B) HeLa cells with stable knockdown of SIRT1 (ShR) or control (ShN) were immunoprecipitated using anti-acetyl-lysine antibody (Ac IP); PABP1 acetylation was blotted with anti-PABP1 antibody. (C) Flag-PABP1 overexpressing 293T cells were lysed and immunoprecipitated using FLAG-M2 beads. The immunoprecipitated complex was incubated with recombinant E.-coli-expressed GST-SIRT1or GST-SIRT1-363A protein in the absence or presence of nicotinamide adenine dinucleotide (NAD) (50 mM) or NAM (200 mM) for 4 hr at 4 C and blotted with anti-acetyl-lysine, anti-Flag, or anti-SIRT1 antibodies. Relative PABP1 acetylation levels were quantified by densitometry of the immunoblots and are presented below the blots. (D) 293T cells transfected with indicated Flag-PABP1 plasmids were lysed and immunoprecipitated using FLAG-M2 beads followed by anti-acetyl-lysine (AcK) western blot. Relative PABP1 acetylation levels were quantified by densitometry of the immunoblots and are presented below the blots. The graph shows quantification of the relative level of PABP1-K95 acetylation from three different experiments; **p < 0.01. Schematic diagram shows K95 of PABP1 is highly conserved. (E) 293T cells transfected with indicated Flag-PABP1 plasmids were immunoprecipitated using FLAG-M2 beads and blotted with anti-PABP1-K95AC antibody. (F) The recombinant His-tagged PABP1 protein purified from E. coli and the Flag-PABP1 protein immunoprecipitated from Flag-PABP1-overexpressing 293T cells were blotted with anti-PABP1-K95AC, anti-pan-Acetyl (Pan-Ac), and anti-PABP1 antibodies. (G and H) 293T cells transfected with indicated plasmids (G) or Flag-PABP1-overexpressing 293T cells treated with NAM (10 mM; 6 hr; H) were lysed and immunoprecipitated with FLAG-M2 beads followed by anti-K95AC western blot. The relative PABP1-K95 acetylation levels in (H) were quantified by densitometry of the immunoblots and are presented below the blots. The graph shows quantification of the relative PABP1-K95AC/PABP1 ratio from three different experiments. (I) HeLa cells transfected with WT or Flag-PABP1 mutants were immunostained with anti-Flag antibody (red) and viewed by fluorescence microscopy (scale bar 20 mm). Protein expression was blotted with anti-Flag antibody. (J) HeLa cells cotransfected with indicated plasmids were immunostained for Flag-SIRT1 (red) and visualized by fluorescence microscopy (scale bar 20 mm). Protein expression was blotted with anti-Flag and anti-GFP antibodies.

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SIRT1 T530A mutant decreased, whereas SIRT1 T530D mutant (mimics phosphorylation) increased, its association with PABP1 (Figure 6E). Moreover, AICAR treatment led to an increment in PABP1-SIRT1 interaction and a concomitant increment in SIRT1 phosphorylation at T530 (Figure 6F), whereas treatment of compound C (AMPK inhibitor) inhibited GD-induced increment of PABP1-SIRT1 association (Figure 6G). These data suggest that AMPK-induced SIRT1 T530 phosphorylation is responsible for GD-induced PABP1-SIRT1 association. Consistently, we observed that AMPKa knockdown blocked GD-induced nuclear accumulation of both PABP1 and poly(A)RNA (Figures 6H and 6I), and the GD-induced increments of SIRT1 T530 phosphorylation (Figure 6J) and SIRT1-PABP1 association (Figure 6K) were markedly inhibited in the AMPK knockdown cells. Collectively, our data suggest that starvation stimulates an AMPKdirected SIRT1 T530 phosphorylation, which causes PABP1SIRT1 association and PABP1 deacetylation, thus leading to nuclear retention of poly(A)RNA. DISCUSSION In this study, we tested the possibility that eukaryotes can shut off protein synthesis via blocking nuclear-cytoplasmic transport of polyadenylated mRNA under starvation conditions. As expected, we observed that energy starvation resulted in marked nuclear accumulation of poly(A)RNA. To our knowledge, this is the first report showing that energy starvation induces a blocking of poly(A)RNA transport. Notably, glucose re-feeding after starvation leads to a recovery of poly(A)RNA nuclear export, indicating eukaryotes adjust its mRNA export according to extracellular glucose levels. Thus, we suggest that the dynamic regulation of poly(A)RNA transport may represent an important strategy by which eukaryotic cells adjust protein synthesis timely according to energy status. In addition, we identify that SIRT1 is required for shutting down poly(A)RNA transport under starvation. Our results show that both overexpression and pharmacologically activation of SIRT1 lead to poly(A)RNA nuclear accumulation. Moreover, SIRT1 depletion leads to marked blocking of starvation-induced poly(A)RNA nuclear accumulation, and SIRT1 is required for fasting-induced poly(A)RNA nuclear accumulation in vivo. Our data therefore reveal a role for SIRT1 in regulating poly(A)RNA transport and suggest that SIRT1 serves as a link between nuclear posttranscriptional modification and cytoplasmic translation in eukaryotes.

As poly(A)RNA-binding proteins, PABPs determine the poly(A)-tail length and assist in mRNA transportation [6, 7]. However, the mechanisms of its transport and its activity in mRNA transport are not fully understood, and sometimes, different reports have conflicting interpretations. Studies in yeast have shown that Pab1 (the major PABP in yeast) is required for the efficient export of mRNA out of the nucleus [5]. Kumar and Glaunsinger suggest that human PABP1 is retained in the cytoplasm via its interactions with RNA [21]. They also proved that a slight elevation of nuclear PABP1 is sufficient to cause poly(A)RNA nuclear accumulation. Khacho et al. demonstrated that eEF1A is involved in PABP1 nuclear export [41]. Woods et al. reported that paxillin associates with PABP1 in the nucleus and plays a role in exporting PABP1 and, by inference, mRNAs [42]. They also proved that a PABP1 mutant which lost paxillin-binding but maintains poly(A)RNA-binding retains in the nucleus. Burgess et al. suggest that nuclear export of PABPs is dependent on ongoing mRNA export [18]. In this study, we show that SIRT1 binds to PABP1 and deacetylates it at K95. Moreover, PABP1 K95 deacetylation causes its nuclear retention, impedes its associations with both poly(A)RNA and translation initiation factors, associating with poly(A)RNA nuclear accumulation, translation reduction, and cell proliferation inhibition. Furthermore, starvation-induced SIRT1 T530 phosphorylation is required for nuclear accumulation of both PABP1 and poly(A) mRNA. These observations lead us to propose a role for SIRT1 as a molecular switch regulating poly(A)RNA nuclear export and translation under normal and stress conditions (Figure 6L). Notably, a report showed that the promoter sequences direct cytoplasmic localization and translation of mRNAs during glucose starvation in yeast [43]. Therefore, our study does not exclude additional mechanisms mediating starvation-induced poly(A)RNA nuclear accumulation. It remains to be clarified whether SIRT1 regulates the functions of PABP1 in mRNA metabolism or the competence of PABP1 to interact with other binding partners. In response to starvation, AMPK is activated [44], which leads to elongation factor 2 phosphorylation and protein synthesis inhibition [45]. AMPK also inhibits 4E-BP1 signaling and mRNA translation via mTOR [46]. Our study reveals that AMPK activation also simultaneously blocks the nucleus mature mRNA export and therefore shuts down the supply of nascent mRNA to the cytoplasm via SIRT1 phosphorylation. Previous study reported that SIRT1 contributes to energy saving via inhibiting rDNA transcription under energy stress conditions [47]. We

Figure 4. PABP1 K95 Deacetylation Impedes Poly(A)RNA Export and Translation (A) HeLa PABP1-knockdown (ShR) cells rescued with PABP1-WT, -K95R, or -K95Q were costained with cy3-oligo(dT)40 (red), anti-PABP1 antibody (green), and DAPI (blue) and visualized by fluorescence microscopy (scale bar 50 mm). Poly(A)RNA nucleo-cytoplasmic (N/C) ratio was quantified. Data show the mean ± SD from three independent experiments. (B) PABP1-knockdown (ShR) and control (ShN) HeLa, HCT116, or U2OS cells were seeded in a 96-well plate (1 3 104/well). Cells were harvested every 24 hr, and the relative cell growth was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Data show the mean ± SD from three independent experiments. PABP1 knockdown efficiency was checked by western blot. (C) PABP1-ShR HeLa cells were recomplemented with RNAi-resistant Flag-tagged PABP1-WT or -K95R. Relative cell growth was determined as described in (B). (D) Cells in (C) were precipitated using poly(U) agarose beads. The coprecipitated PABP1s were blotted with anti-Flag antibody. The relative PABP1/RNA ratio was analyzed as in Figure 2G. (E) 293T cells transfected with indicated plasmids were lysed and immunoprecipitated using FLAG-M2 beads followed by anti-GFP and anti-Flag western blot. (F) PABP1-ShR HeLa cells rescued with RNAi-resistant Flag-tagged PABP1-WT or -K95R were labeled with 35S-methionine-protein labeling mix (20 mCi/mL) for 1 hr. Radioactivity incorporation of newly synthesized proteins was analyzed by SDS-PAGE and visualized by autoradiography (upper panel). Cell lysate was analyzed by western blot using anti-actin and anti-PABP1 antibodies (lower two panels).

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show that the PABP1 knockdown cells recomplemented with PABP1-K95Q have increased rate of ATP consumption as compared to normal or PABP1-WT rescued cells, suggesting that shutting off poly(A)RNA nuclear-cytoplasmic transport also contributes to energy saving upon starvation. These findings advance our understanding of how eukaryotes coordinate multiple pathways to effectively control translation to maintain energy homeostasis under starvation conditions. Collectively, our observations indicate that SIRT1 forms a complex with PABP1 and dynamically regulates the on/off of polyadenylated RNA nuclear-cytoplasmic transport and thus adjusts protein synthesis to maintain energy homeostasis according to energy status. The starvation-induced blocking of poly(A) RNA nuclear export supports the idea that the poly(A)-tail of RNA also provides a basis for eukaryotes to effectively shut down mature mRNA export to tailor protein synthesis in response to energy shortage. Previous studies have revealed that both heat and ethanol shock cause nuclear accumulation of poly(A)RNA in yeast [48, 49]. In this study, we also observed an increased SIRT1-PABP1 interaction in cells exposed to DNA-damaging drugs, including etoposide and MNNG treatments, and these stresses also caused a marked nuclear accumulation of PABP1 and poly(A)RNA (Figure S5), suggesting that the eukaryotic SIRT1-PABP1 complex represents a general sensor for fine tuning of RNA transport under stress conditions. Of note, polyadenylation is not only involved in RNA transport and translation but also in mRNA stability, and PABP1 also has other roles in mRNA-specific translational regulation and nonsense-mediated decay [20, 50]. Moreover, many eukaryotic non-coding RNAs are always polyadenylated. Further study on the functions of SIRT1 in RNA metabolism will expand our understanding of how eukaryotes maintain or re-establish cellular homeostasis under diverse stress conditions. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B Cell Culture METHOD DETAILS

B

Plasmids Phosphorylation/Acetylation Specific Antibodies B Immunoprecipitation and In Vitro Binding B Immunofluorescence Staining B Immunohistochemical Staining B Fluorescence In Situ Hybridization B Measurement of ATP Level B Poly(U) Pull Down Assay B In Vitro Deacetylation Assay B Protein Synthesis Assay B Cell Growth Assay B Cell Size Assay B Nucleocytoplasmic Separation QUANTIFICATION AND STATISTICAL ANALYSES B Quantitation of Poly(A)RNA Nucleo-cytoplasmic Ratio B Statistical Analysis B

d

SUPPLEMENTAL INFORMATION Supplemental Information includes five figures and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2017.06.040. AUTHOR CONTRIBUTIONS P.S., G.F., L.S., J.L., W.W., C.H., and X.S. performed experiments. X.Z., Q.Z., L.C., and Y.C. provided critical reagents. S.Z. and C.W. interpreted data and wrote the manuscript. ACKNOWLEDGMENTS This work was supported by the National Key Research Program of China (2016YFC1304800), NSFC (81372146, 31671462, 31671433, and 81602616), a Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant (20152524), and the Shanghai Municipal Science and Technology Commission (16YF1409100 and 17YF1415500). Received: September 29, 2016 Revised: May 4, 2017 Accepted: June 15, 2017 Published: July 27, 2017 REFERENCES 1. Hunt, A.G., Xu, R., Addepalli, B., Rao, S., Forbes, K.P., Meeks, L.R., Xing, D., Mo, M., Zhao, H., Bandyopadhyay, A., et al. (2008). Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling. BMC Genomics 9, 220. 2. Lei, E.P., and Silver, P.A. (2002). Protein and RNA export from the nucleus. Dev. Cell 2, 261–272.

Figure 5. Starvation Induces SIRT1-Dependent PABP1 Deacetylation and Nuclear Accumulation (A) HeLa cells treated with or without GD (12 hr) were fixed and costained for PABP1 (green) and DNA (blue) and visualized by fluorescence microscopy (scale bar 20 mm). (B) 293T cells treated with or without GD (4 hr) were lysed, immunoprecipitated using anti-PABP1 antibody, and blotted for SIRT1 and PABP1. (C) SIRT1 stable knockdown (ShR) or vector control (ShN) HeLa cells treated with or without GD (4 hr) were lysed, immunoprecipitated using anti-PABP1 antibody, and blotted for PABP1-K95AC. Relative PABP1-K95 acetylation levels were quantified by densitometry of the immunoblots and are presented below the blots. The graph shows quantification of the relative PABP1-K95AC/PABP1 ratio from three different experiments; **p < 0.01. (D) HeLa cells rescued with PABP1-WT or -K95Q were treated with GD and costained with cy3-oligo(dT)40 (red), anti-PABP1 antibody (green), and DAPI (blue) and visualized by fluorescence microscopy (scale bar 50 mm). (E) Images showing immunohistochemical staining of PABP1and PABP1-K95AC in the cerebral cortex of WT and SIRT1-KO mice under normal feeding or 24-hr fasting (scale bar 50 mm). For negative control, the primary antibody was replaced by PBS. (F) Indicated PABP1 knockdown and rescued stable cell lines were treated with GD for the indicated time periods, and the relative intracellular ATP levels were analyzed. All experiments were repeated three times; data represent mean ± SD; *p < 0.05; **p < 0.01. See also Figure S4.

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3. Sachs, A.B., Davis, R.W., and Kornberg, R.D. (1987). A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7, 3268–3276.

16. Kahvejian, A., Roy, G., and Sonenberg, N. (2001). The mRNA closed-loop model: the function of PABP and PABP-interacting proteins in mRNA translation. Cold Spring Harb. Symp. Quant. Biol. 66, 293–300.

4. Gray, N.K., Coller, J.M., Dickson, K.S., and Wickens, M. (2000). Multiple portions of poly(A)-binding protein stimulate translation in vivo. EMBO J. 19, 4723–4733.

17. Ma, S., Bhattacharjee, R.B., and Bag, J. (2009). Expression of poly(A)binding protein is upregulated during recovery from heat shock in HeLa cells. FEBS J. 276, 552–570.

5. Brune, C., Munchel, S.E., Fischer, N., Podtelejnikov, A.V., and Weis, K. (2005). Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export. RNA 11, 517–531.

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6. Eliseeva, I.A., Lyabin, D.N., and Ovchinnikov, L.P. (2013). Poly(A)-binding proteins: structure, domain organization, and activity regulation. Biochemistry (Mosc.) 78, 1377–1391. 7. Mangus, D.A., Evans, M.C., and Jacobson, A. (2003). Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol. 4, 223. 8. Deo, R.C., Bonanno, J.B., Sonenberg, N., and Burley, S.K. (1999). Recognition of polyadenylate RNA by the poly(A)-binding protein. Cell 98, 835–845. 9. Hosoda, N., Lejeune, F., and Maquat, L.E. (2006). Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails. Mol. Cell. Biol. 26, 3085–3097. 10. Bushell, M., Wood, W., Carpenter, G., Pain, V.M., Morley, S.J., and Clemens, M.J. (2001). Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages. J. Biol. Chem. 276, 23922–23928. 11. Afonina, E., Stauber, R., and Pavlakis, G.N. (1998). The human poly(A)binding protein 1 shuttles between the nucleus and the cytoplasm. J. Biol. Chem. 273, 13015–13021. 12. Wells, S.E., Hillner, P.E., Vale, R.D., and Sachs, A.B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Mol. Cell 2, 135–140. 13. Jackson, R.J., Hellen, C.U.T., and Pestova, T.V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127. 14. Munroe, D., and Jacobson, A. (1990). mRNA poly(A) tail, a 30 enhancer of translational initiation. Mol. Cell. Biol. 10, 3441–3455. 15. Tarun, S.Z., Jr., and Sachs, A.B. (1995). A common function for mRNA 50 and 30 ends in translation initiation in yeast. Genes Dev. 9, 2997–3007.

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Figure 6. AMPK Triggers PABP1 and Poly(A)RNA Redistribution via Phosphorylating SIRT1 (A and B) HeLa cells treated with or without AICAR (200 mM; 12 hr) were either immunostained for PABP1 (green; A) or probed with cy3-oligo(dT)40 (red; B) and visualized by fluorescence microscopy (scale bar 20 mm). Nuclei (blue) were stained with DAPI. (C) 293T cells treated with GD for indicated times were lysed and examined by western blot using antibodies against SIRT1 phosphorylation at S14, T530, and T719, respectively. (D) 293T cells transfected with indicated plasmids were treated with or without GD (4 hr); cell lysates were immunoprecipitated using FLAG-M2 beads followed by western blot analysis. (E) 293T cells transfected with indicated plasmids were lysed and immunoprecipitated using FLAG-M2 beads followed by western blot analysis. (F) 293T cells treated with AMPK activator AICAR (200 mM; 0–4 hr) were lysed and immunoprecipitated with anti-SIRT1 antibody followed by western blot analysis. (G) 293T cells were treated with AMPK inhibitor compound C (10 mM; 1 hr) followed by 4-hr GD treatment. Cell lysates were immunoprecipitated with anti-SIRT1 antibody, and coprecipitated PABP1s were determined by western blot. Coprecipitated PABP1s were quantified by densitometry of bands. Bar graph shows quantification of relative PABP1-SIRT1 association. (H and I) HeLa cells with stable knockdown of AMPKa (ShAMPK) or a vector control (ShN) treated with or without GD (12 hr) were either immunostained for PABP1 (green; H) or probed with cy3-oligo(dT)40 (red; I) and visualized by fluorescence microscopy (scale bar 20 mm). Nuclei (blue) were stained with DAPI. (J) Indicated cell lines were treated with GD (0–4 hr) and subsequently lysed followed by western blotting. (K) Indicated cell lines were treated with or without GD (4 hr) followed by immunoprecipitation with anti-SIRT1 antibody and western blot analysis. (L) A model showing how eukaryotic cells sense the energy status and redistribute mature mRNA to match the environmental demand. Under normal conditions, cells maintain basal SIRT1-T530 phosphorylation at a relatively low level, which has weak effects on PABP1-K95 acetylation and nuclear PABP1-poly(A)mRNA association, thus allowing normal mature mRNA nuclear export and cytoplasmic translation; whereas upon starvation, activated AMPK phosphorylates SIRT1T530, promotes nuclear SIRT1-PABP1 association and PABP1-K95 deacetylation, and displaces poly(A)mRNA from PABP1, leading to shutting down mature mRNA nuclear export and protein synthesis, thereby maintaining cellular energy homeostasis under energy stress conditions. See also Figure S5.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT OR RESOURCE

SOURCE

IDENTIFIER

Mouse monoclonal anti-PABP1

Santa Cruz

Cat# SC-32318; RRID: AB_628097

Rabbit polyclonal anti-SIRT1

Santa Cruz

Cat# SC-15404; RRID: AB_2188346

Rabbit polyclonal anti-GFP

Santa Cruz

Cat# SC-8334

Antibodies

Mouse monoclonal anti-Flag

Sigma

Cat# F1804; RRID: AB_262044

Mouse monoclonal anti-b-Actin

Sigma

Cat# A5316; RRID: AB_476743

Mouse monoclonal anti-GST

Proteintech

Cat# 66001-1-Ig; RRID: AB_10951482

Mouse monoclonal anti-His

Proteintech

Cat# 66005-1-Ig; RRID: AB_11232599

Anti-acetyl lysine antibody

Cell Signaling Technology

Cat# 9441S; RRID: AB_331805

Rabbit polyclonal anti-AMPK

Bioworld

Cat# BS1009; RRID: AB_1663336

Rabbit polyclonal anti-SIRT1 S14 phosphorylation antibody

[40]

N/A

Rabbit polyclonal anti-SIRT1 T530 phosphorylation antibody

[40]

N/A

Rabbit polyclonal anti-SIRT1 T719 phosphorylation antibody

[40]

N/A

Rabbit polyclonal anti-PABP1 K95 acetylation antibody

This paper

N/A

Bacterial and Virus Strains Trans5a Chemically Competent Cell

TransGen Biotech

Cat# CD201

E.coli. BL21

Thermo Scientific

Cat# C600003

Nicotinamide

Sigma

Cat# 72340

Resveratrol

Sigma

Cat# R5010

2-DG

Sigma

Cat# D8375

AICAR

Sigma

Cat# A8129

Compound C

Sigma

Cat# P5499

Nocodazole

Sigma

Cat# M1404

NAD+

Sigma

Cat# N8285

RNase I

Invitrogen

Cat# AM2294

RNase A

Thermo Fisher

Cat# EN0531

IPTG

Thermo Scientific

Cat# 15529019

Ribonucleoside vanadyl complexes

Sigma

Cat# R3380

RNasin Ribonuclease Inhibitors

Promega

Cat# N2511 N/A

Chemicals, Peptides, and Recombinant Proteins

Biotin-50 -Oligo(dT)40 Probe

Bioligo

Cy3-Conjugated Oigo(dT)40 Probe

Life technologies

N/A

ANTI-FLAG M2 Affinity Gel

Sigma

Cat# A2220

Glutathione-Sepharose 4B Resin

Solarbio

Cat# P2020-5

Ni-NTA His.Bind Resin

Novagen

Cat# 70666-4

Sulfolink Coupling Resin

Thermo Scientific

Cat# 20402

Poly(U) Agarose

Sigma

Cat# P8563

Streptavidin-HRP

ThermoFisher Scientific

Cat# N100

PMSF Protease Inhibitor

Thermo Fisher

Cat# 36978

MTT

Sigma

Cat# M2128

EasyTag Express Protein Labeling Mix [35S]-

PerkinElmer

Cat# NEG772

Flag Peptide

Sigma

Cat# F3290 (Continued on next page)

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Continued REAGENT OR RESOURCE

SOURCE

IDENTIFIER

Non-Acetylated Peptide (CRDPSLRKSGV)

GL Biochem

N/A

Recombinant Protein:His-PABP1

This paper

N/A

Recombinant Protein:GST–SIRT1

This paper

N/A

Recombinant Protein:GST–SIRT1-363A

This paper

N/A

TIANprep Mini Plasmid Kit

TIANGED

Cat# DP103-03

Quik-Change Site-Directed Mutagenesis Kit

Stratage

Cat# 200519

ATP assay kit

Beyotime

Cat# S0026

HeLa

ATCC

RRID: CVCL_0030

HCT116

ATCC

RRID: CVCL_0291

HEK293T

ATCC

RRID: CVCL_0063

U2OS

ATCC

RRID: CVCL_0042

SIRT1-WT and SIRT1-KO MEF cells

This paper

N/A

PABP1-knockdown stable cell lines

This paper

N/A

SIRT1-knockdown stable cell lines

This paper

N/A

AMPK-knockdown stable cell lines

This paper

N/A

PABP1 knockdown-rescue stable cell lines

This paper

N/A

Dr. Michael McBurney, University of Ottawa, Canada

N/A

PABP1 siRNA (GGACAAATCCATTGATAATAA)

This paper

N/A

AMPK siRNA (ATGATGTCAGATGGTGAATTT)

This paper

N/A

SIRT1 siRNA (AGCGATGTTTGATATTGAA)

[30]

N/A

Plasmid: Flag-PABP1

This paper

N/A

Plasmid: GFP-PABP1

This paper

N/A

Plasmid: His-PABP1

This paper

N/A

Plasmid: Flag-PABPN1

This paper

N/A

Plasmid: Flag–SIRT1

This paper

N/A

Plasmid: Flag–SIRT1-363A

This paper

N/A

Plasmid: GFP–SIRT1

This paper

N/A

Plasmid: GST–SIRT1

This paper

N/A

Plasmid: GST–SIRT1-363A

This paper

N/A

Plasmid: Flag-PABP1 deletion mutants

This paper

N/A

Plasmid: Flag-PABP1 deletion mutants

This paper

N/A

Plasmid: Flag-PABP1-WT, K95R and K95Q

This paper

N/A

Plasmid: Flag-SIRT1-719A, 530A and 530D

[40]

N/A

Plasmid: GFP-eIF4G

This paper

N/A

Plasmid: GFP-eIF4B

This paper

N/A

ImageJ

NIH

https://imagej.nih.gov/ij/

Image-Pro Plus

[51, 52]

N/A

Cell Quest software

[53]

N/A

Critical Commercial Assays

Experimental Models: Cell Lines

Experimental Models: Organisms/Strains SIRT1 knockout mouse 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, Chuangui Wang ([email protected]). e2 Current Biology 27, 1–14.e1–e5, August 7, 2017

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EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice SIRT1+/ mice were a kind gift from Michael McBurney, University of Ottawa, Canada. All experiments were conducted in male SIRT1+/+ and SIRT1/ littermates. Mice were housed in a specific pathogen-free facility on a 12h light-dark cycle with a libitum access to food and water. For the fasting experiments, 4-5-month old SIRT1-WT and -KO mice were fed either with a standard CD or fasted for a period of 24h but had free access to water. Animal use and care protocols, including all operation procedures, were approved by the Institutional Animal Care and Use Committee of East China Normal University. Cell Culture HeLa (Female), HCT116 (Male), HEK293T (Female) and U2OS (Female) cancer cell lines were obtained from the American Type Culture Collection. These cell lines have been authenticated using STR profiling at Beijing HAKE Genetics Co., Ltd., which showed 100% matching to the HeLa, HCT116, HEK293T and U2OS cell lines found in ATCC data bank. SIRT1-WT and SIRT1-KO MEF cells were derived from SIRT1-WT and -KO mice. All cells were maintained in DMEM with 10% (v/v) fetal bovine serum. METHOD DETAILS Plasmids SIRT1-knockdown plasmids were previously generated [30]. PABP1-knockdown plasmids were generated by inserting CCGGGGA CAAATCCATTGATAATAACTCGAG TTATTATCAA TGGATTTGTCCTTTTTTG into the AgeI and EcoRI restriction sites of pLKO.1. PABP1-knockdown or vector control stable cell lines were established by transient transfection and selected with puromycin. To construct the PABP1 knockdown-rescue system, RNAi-resistant PABP1-WT, K95R and K95Q expression plasmids were generated using the following primer: TAGGCAACATA TTCATTAAAAATCTGG ACAAGAGTATCGACAA TA AAGCA CTGTATGATACA (mutated nucleotides underlined). PABP1 knockdown-rescue stable cell lines were established by transfection of RNAi-resistant PABP1 expressing plasmid and selection with hygromycin. AMPK-knockdown plasmids were generated by inserting CCGGATGATGTCA GATGGTGAATTTCTCGAGAAATTCACCATCTCACATCATTTTTTTG into the AgeI and EcoRI restriction sites of pLKO.1. The human PABP1 expression plasmid was constructed by inserting the 1911bp coding region of the PABP1 gene into pCDNA3.0-Flag, pCDNA3.0-GFP, and PGEX4T-1 vectors. PABP1 deletion mutants were constructed by PCR from Flag -PABP1. PABP1 and SIRT1 mutations were generated using site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis Kit, Stratage). GFPeIF4G and GFP-eIF4B expression plasmids were constructed by inserting the coding regions into pCDNA3.0-GFP vector respectively. Phosphorylation/Acetylation Specific Antibodies Two rabbit antiserums against SIRT1 phosphorylated at S14, T530 and T719 were raised as previously described [40]. A rabbit antiserum against PABP1 K95 acetylation was raised using peptide (CRDPSLR K[Ac] SGV) of which K95 is acetylated and indicated as K [Ac]. The antiserum was pre-cleaned by affinity chromatography using the corresponding non-acetylated peptide (CRDPSLRKSGV) coupled to Sulfolink coupling resin (Thermo Scientific) and purified by affinity chromatography. Immunoprecipitation and In Vitro Binding Cell lysate preparation and protein immunoprecipitation were performed as previously described [40]. For in vitro binding assay, GST-fusion and His-fusion constructs were introduced into E. coli BL21 (DE3) and the fusion protein were induced with 0.1-1mM IPTG for 3-8h at 18 C. The induced proteins were purified using glutathione-Sepharose 4B resin or Ni-NTA agarose beads. For binding assay, GST or GST-SIRT1 were incubated with His-PABP1 in binding buffer (100mM NaCl, 50mM Tris HCl pH7.5, 15mM EGTA, 1mM DTT, and 1mM PMSF, 0.2% Triton X-100, 10 mg/ml RNase A) for 2h at 4 C. Protein complex was pulled down with glutathioneagarose beads for 2h at 4 C, washed and subjected to western blot analysis. Immunofluorescence Staining Immunofluorescence was operated as previously described [54]. Briefly, cells were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS, incubated for 30 min in permeabilization buffer (0.1% Triton X-100 in PBS), block with 2% BSA, and incubated with primary antibody for 2h at room temperature or overnight at 4 C followed by incubation with fluorescently conjugated secondary antibody for 2h at room temperature. Immunohistochemical Staining Immunohistochemical staining in tissues was performed using anti-PABP1 antibody and visualized as previously described [40]. Antibodies against PABP1, PABP1-K95AC or SIRT1T530P were used for immunohistochemistry staining. In situ hybridization of poly(A) RNA in tissues was performed by using biotin-50 -oligo(dT)40 (Bioligo) probe and operated as previously described [55]. Briefly, fixed dehydrated tissues were rehydrated and washed twice for 5 min with PBS, incubated with 10 mg/ml Pepsin for 3 min at 37 C, washed twice with PBS, balanced with 2 3 SSC for 15 min then pre-hybridized with hybridization buffer (50% formamide, 10% dextran sulfate, 0.02% BSA, 200mg Escherichia coh tRNA in 2 3 SSC) for 2h at 37 C, and incubated with biotin-50 -oligo(dT)40 (Bioligo) probe in Current Biology 27, 1–14.e1–e5, August 7, 2017 e3

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hybridization buffer overnight at 60 C. Tissues were then washed twice for 30 min at 60 C with 2 3 SSC and 0.1 3 SSC respectively and incubated with block buffer (5% BSA in 2 3 SSC) for 1h at room temperature, processed with Streptavidin-HRP (N100, ThermoFisher Scientific) in hybridization buffer for 1h at room temperature, followed by microscopy analysis. Quantitation of nuclear versus cytoplasm intensity of poly(A)RNA in Lung and Colon tissues were analyzed by Image-Pro Plus software as described previously [51, 52]. Fluorescence In Situ Hybridization Poly(A)RNA fluorescence in situ hybridization assay was performed as described previously [18] using a Cy3-conjugated oligo(dT)40 probe (Life technologies, Shanghai). To analyze the poly(A)RNA subcellular localization under starvation, we generated mouse embryonic fibroblasts (MEFs) from 13.5-day-old embryos. MEFs at passages 3-4 were used. Measurement of ATP Level ATP levels were measured using ATP assay kit (Beyotime). Poly(U) Pull Down Assay Poly(U)-Pull Down assay was performed as previously described [56]. Cells were lysed in lysis buffer (0.1M NaCl, 10mM MgCl2, 0.3M Tris-Cl PH 7.5, 1mM DTT, 10mM Ribonucleoside vanadyl complexes, 30U/ml RNasin Ribonuclease inhibitor, 1% Triton X-100). Of note, the Ribonucleoside vanadyl complexes and RNasin Ribonuclease inhibitors are omitted when RNase A is used. Cell extracts were centrifuged at 10,000 g for 5 min and the insoluble debris were discarded. Before poly(U)-pull down assay, dry poly(U) agarose (Sigma) was hydrated in 0.1M NaCl and 10mM Tris-HCl (PH 7.4) at room temperature, then washed in elution buffer (0.1M NaCl, 0.01M EDTA, 0.5M Tris-HCl at PH 7.4, 0.2% SDS, 25% formamide) and high salt binding buffer (0.7M NaCl, 0.01M EDTA, 0.5M Tris-HCl PH 7.4, 0.2% lauryl sarcosine, 12% formamide) one time each for 5 min at 70 C. Then the pretreated poly(U) agaroses were added to cell lysates, rotated for 2h at 4 C, then 80% beads were eluted by boiling in 1 x SDS-PAGE sample buffer for western blot analysis, and the left 20% beads were eluted in 1mM EDTA (PH 8.0) to examine the total amount of mRNA precipitated by the poly(U) beads. The intensity of PABP1 western blot band was normalized to the level of mRNA in each sample. In Vitro Deacetylation Assay In vitro deacetylation was performed as previously described [57]. Briefly, 293T cells overexpressing Flag-PABP1 was lysed and immunoprecipitated with anti-FLAG M2 agarose (Sigma) and the enriched PABP1 proteins were eluted with FLAG peptide. The eluted proteins were incubated with GST-SIRT1 or GST-SIRT1-363A in 30 mL of deacetylation buffer (50mM Tris-HCl, PH 9.0, 50mM NaCl, 4mM MgCl2, 0.5mM DTT, 0.1mM PMSF, 5% glycerol, 0.02% NP-40, and protease inhibitors) in the presence or absence of 50 mM NAD+ or 200 mM NAM for 2h at 30 C. The reactions were subjected to SDS-PAGE and analyzed by western Blot. Protein Synthesis Assay Cells seeded in 6-well plates were washed twice with PBS, incubated with methionine-deleted medium for 1h, replace medium with methionine-free DMEM containing 35S-protein labeling mix (20 mCi/mL) for 1h. Then cells were washed with cold PBS, lysed in lysis buffer and subjected to SDS-PAGE, labeled proteins were visualized by autoradiography. Cell Growth Assay Cell growth assay was operated as previously described [58]. Briefly, cells were harvested every 24 hr, the MTT solution was added for 4 hr, then the reactions were stopped by addition of DMSO solution and the samples were measured at 490 nm. Cell Size Assay To determine cell size, FACS analysis with Cell Quest software was performed as previously described [53]. In brief, HeLa cells cultured under energy starvation for the indicated times were harvested, fixed in 70% (v/v) ice cold ethanol, and followed by PI staining. Nucleocytoplasmic Separation Cells were lysed by the CLB buffer (10mM HEPES, 10mM KH2PO4, 5mM NaHCO3, 5mM EDTA, 1mM CaCl2, 0.5mM MgCl2) for 10 min on ice, homogenized by 15 strokes with 1 mL needle, then sucrose was added to reach a final concentration of 0.25M followed by centrifugation at 6300 g for 5 min at 4 C. The supernatant (the cytoplasmic fraction) were kept. The pellet (containing the nuclear fraction) was washed once with TSE buffer (10mM Tris, 300mM sucrose, 1mM EDTA, 0.1% NP-40, PH 7.5), then lysed with 1x SDS-PAGE sample buffer and boiled for 10 min. QUANTIFICATION AND STATISTICAL ANALYSES Quantitation of Poly(A)RNA Nucleo-cytoplasmic Ratio Quantitation of nuclear versus cytoplasm intensity of poly(A)RNA in cells was performed as described previously [59]. Briefly, images were exported as Tiff files and fluorescence intensities in the nuclear and cytoplasmic regions were quantified using ImageJ. e4 Current Biology 27, 1–14.e1–e5, August 7, 2017

Please cite this article in press as: Shan et al., SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport, Current Biology (2017), http://dx.doi.org/10.1016/j.cub.2017.06.040

Nucleo-cytoplasmic (N/C) ratio was calculated from fluorescence intensity of the nucleus normalized to the cytoplasm of each cell, the mean N/C ratios were calculated from mean N/C ratio measured of 50 cells for a given experiment. Statistical Analysis All experiments were structured with a control group and experimental groups, and repeated at least three times. All the samples analyzed were included in quantification. All statistical analyses were performed using two-tailed Student’s t tests in Microsoft Excel without testing for whether assumptions for normality were met due to low sample sizes. The results are expressed as mean ± standard deviation (SD) as indicated in the figure legends. The ‘‘n’’ represents the number of cells or experimental replications as indicated in the figure legends. The p values are also indicated in the figure legends where relevant, and p < 0.05 were considered significant.

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