Hepatic Posttranscriptional Network Comprised of CCR4–NOT Deadenylase and FGF21 Maintains Systemic Metabolic Homeostasis

Hepatic posttranscriptional network comprised of CCR4–NOT deadenylase and FGF21 maintains systemic metabolic homeostasis

Masahiro Moritaa,b,c,1,2, Nadeem Siddiquid,e,1, Sakie Katsumuraa,b, Christopher Rouyad,e, Ola Larssonf, Takeshi Nagashimag, Bahareh Hekmatnejadh,i, Akinori Takahashij, Hiroshi Kiyonarik, Mengwei Zanga,b, René St-Arnaudh,i, Yuichi Oikel, Vincent Giguèred,e,m, Ivan Topisirovicd,m,n, Mariko Okada-Hatakeyamag,o, Tadashi Yamamotoj,2, and Nahum Sonenbergd,e,2

aDepartment of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; bBarshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; cInstitute of Resource Development and Analysis, Kumamoto University, 860-0811 Kumamoto, Japan; dDepartment of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; eGoodman Cancer Research Centre, McGill University, Montreal, QC H3A 1A3, Canada; fDepartment of Oncology-Pathology, Scilifelab, Karolinska Institutet, SE-171 76 Stockholm, Sweden; gLaboratory for Integrated Cellular Systems, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045 Kanagawa, Japan; hResearch Centre, Shriners Hospital for Children–Canada, Montreal, QC H4A 0A9, Canada; iDepartment of Human Genetics, McGill University, Montreal, QC H3A 2T5, Canada; jCell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 904-0495 Okinawa, Japan; kLaboratories for Animal Resource Development and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, 650-0047 Hyogo, Japan; lDepartment of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, 860-8556 Kumamoto, Japan; mGerald Bronfman Department of Oncology, McGill University, Montreal, QC H2W 1S6, Canada; nLady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada; and oLaboratory of Cell Systems, Institute for Protein Research, Osaka University, Suita, 565-0871 Osaka, Japan

Contributed by Nahum Sonenberg, February 24, 2019 (sent for review September 17, 2018; reviewed by Jack D. Keene and David J. Mangelsdorf) Whole-body metabolic homeostasis is tightly controlled by hormone- CCR4–NOT-dependent deadenylation by RBPs and the miRISC like factors with systemic or paracrine effects that are derived (18). However, the composition and function of CCR4–NOT from nonendocrine organs, including adipose tissue (adipokines) containing messenger ribonucleoprotein (mRNP) complexes in and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is a

physiological and pathological states remain obscure (19). MEDICAL SCIENCES hormone-like protein, which is emerging as a major regulator of The CCR4–NOT complex has been implicated in the develop- whole-body metabolism and has therapeutic potential for treating ment of metabolic diseases (20–24). These disorders, including di- metabolic syndrome. However, the mechanisms that control FGF21 abetes, steatosis, hyperlipidemia, and obesity, are major worldwide levels are not fully understood. Herein, we demonstrate that FGF21 production in the liver is regulated via a posttranscriptional network consisting of the CCR4–NOT deadenylase complex and RNA-binding Significance protein tristetraprolin (TTP). In response to nutrient uptake, CCR4– NOT cooperates with TTP to degrade AU-rich mRNAs that encode The mRNA poly(A) tail controls gene expression at post- pivotal metabolic regulators, including FGF21. Disruption of CCR4– transcriptional levels, including mRNA degradation and trans- NOT activity in the liver, by deletion of the catalytic subunit CNOT6L, lation. Here, we show that a hitherto unknown hepatic increases serum FGF21 levels, which ameliorates diet-induced meta- posttranscriptional network centered on the CCR4–NOT dead- bolic disorders and enhances energy expenditure without disrupting enylase plays a seminal role in regulating FGF21 expression and bone homeostasis. Taken together, our study describes a hepatic its effects on systemic metabolism. A genome-wide search for CCR4–NOT/FGF21 axis as a hitherto unrecognized systemic regula- CNOT6L-associated mRNAs unveiled the mechanism whereby tor of metabolism and suggests that hepatic CCR4–NOT may serve CNOT6L selectively degrades a subset of mRNAs encoding met- as a target for devising therapeutic strategies in metabolic syndrome abolic factors, including FGF21. Disruption of CCR4–NOT dead- and related morbidities. enylase activity, by targeting its catalytic subunit CNOT6L, leads to an increase in FGF21 levels, which is paralleled by a dramatic CCR4–NOT | deadenylase | FGF21 | hepatokine | metabolic syndrome improvement of metabolic syndrome. Overall, our findings describe a new paradigm in regulation of whole-body metab- he mRNA poly (A) tail plays an essential role in post- olism, whereby a hepatic posttranscriptional network governs Ttranscriptional regulation of gene expression by affecting systemic metabolic regulation via FGF21. mRNA decay and translation (1–3). Deadenylation is the rate-limiting step in mRNA degradation that, together with transcription, de- Author contributions: M.M., N. Siddiqui, T.Y., and N. Sonenberg designed research; M.M., N. Siddiqui, S.K., C.R., B.H., A.T., and H.K. performed research; M.M., O.L., H.K., M.Z., termines steady-state mRNA levels (4). mRNA deadenylation is R.S.-A., Y.O., V.G., and M.O.-H. contributed new reagents/analytic tools; M.M., O.L., primarily catalyzed by the CCR4–NOT complex, a multisubunit T.N., and B.H. analyzed data; and M.M., N. Siddiqui, S.K., C.R., O.L., I.T., M.O.-H., T.Y., protein machinery composed of the CCR4 (CNOT6L/CNOT6) and N. Sonenberg wrote the paper. deadenylase, the CNOT1 scaffold protein, and several regu- Reviewers: J.D.K., Duke University; and D.J.M., The University of Texas Southwestern latory proteins (CNOT2–CNOT11) (5–7). Medical Center. Direct recruitment of the CCR4–NOT complex to target The authors declare no conflict of interest. mRNAs destined for deadenylation and decay is mediated by sev- Published under the PNAS license. eral RNA-binding proteins (RBPs), including tristetraprolin (TTP), Data deposition: The data reported in this paper have been deposited in the Gene Ex- – pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. Nanos2, and Roquin (8 13). In addition, posttranscriptional si- GSE62365). – lencing by miRNAs occurs through association of the CCR4 NOT 1M.M. and N. Siddiqui contributed equally to this work. complex with the miRNA-induced silencing complex (miRISC) 2 To whom correspondence may be addressed. Email: [email protected], tadashi. (14–16). The selectivity of mRNA deadenylation is controlled by [email protected], or [email protected]. – cis-acting mRNA elements to which CCR4 NOT-associated RBPs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and miRISC bind (17, 18). Previous structural and biochemi- 1073/pnas.1816023116/-/DCSupplemental. cal studies have provided mechanistic models for the selective Published online March 29, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1816023116 PNAS | April 16, 2019 | vol. 116 | no. 16 | 7973–7981 Downloaded by guest on September 27, 2021 health problems causally associated with dysregulation of meta- the stability of mRNAs that contain AREs and are enriched in bolic homeostasis. Whole-body metabolic homeostasis is closely those encoding metabolic regulators. controlled in a systemic or paracrine manner by hormone-like factors secreted from nonendocrine organs, such as adipose TTP Recruits the CCR4–NOT Complex to ARE-Containing Target mRNAs tissue (adipokines) and liver (hepatokines) (25, 26). Hormone- Destined for Degradation. Because the CCR4–NOT complex di- like proteins can either enhance [e.g., fibroblast growth factor 21 rectly interacts with the ARE-binding protein, TTP (9, 11, 12, 36), (FGF21) and leptin] or impair (e.g., resistin and selenoprotein P) we investigated whether TTP promotes CCR4–NOT-dependent energy metabolism (26, 27). However, there are no studies that degradation of endogenous target mRNAs in hepatocytes. The directly link the deadenylase activity of CCR4–NOT to hormone- CCR4–NOT complex subunits (CNOT6L, CNOT1, CNOT3, like proteins and metabolic disorders. and CNOT7) were precipitated with TTP from a hepatocyte Here, we identified target mRNAs associated with the extract (Fig. 2A). TTP directly binds to CNOT1 via a conserved CNOT6L deadenylase subunit of the CCR4–NOT complex in phenylalanine (F319) (9). Mutation of F319 to alanine (F319A) the liver by performing RNA immunoprecipitation followed by dramatically reduced the interaction between TTP and the microarray analysis (RIP-CHIP). We demonstrate that, in re- CCR4–NOT complex subunits (Fig. 2A). Moreover, CNOT6L sponse to feeding, the CCR4–NOT/TTP complex targets the coimmunoprecipitated with TTP in hepatocytes (Fig. 2B). De- AU-rich mRNA encoding the hepatokine FGF21, which allevi- pletion of TTP (Fig. 2C and SI Appendix, Fig. S3A), but not – ates diet-induced metabolic disorders (28–31). Deletion of the another CCR4 NOT-associated protein, Roquin (SI Appendix, – Cnot6l gene in mice decreased susceptibility to diet-induced Fig. S3 B D), impaired the binding of CNOT6L to ARE- metabolic disorders, such as obesity, steatosis, and hyperlipid- containing mRNAs (Fig. 2D), and enhanced their stability and emia in a deadenylase activity-dependent manner. We found that steady-state levels (Fig. 2 E and F). These results demonstrate – the observed metabolic disorders can largely be explained by that TTP mediates the interaction of the CCR4 NOT complex CNOT6L-dependent control of Fgf21 mRNA decay. Thus, we with ARE-containing mRNAs encoding several pivotal meta- conclude that the CNOT6L deadenylase targets a subset of bolic regulators to promote their degradation. mRNAs, including Fgf21, to control whole-body metabolism. The CCR4–NOT/TTP Complex Degrades the Hepatokine Fgf21 mRNA in Our findings show that CNOT6L plays a major role in regulation of Response to Feeding. Fgf21 mRNA contains canonical AREs in its FGF21 levels, thus providing unprecedented evidence that CNOT6L 3′UTR (SI Appendix, Fig. S3 E and F) and was dramatically may serve as a therapeutic target to treat metabolic diseases. − enriched (P = 4.7 × 10 5) in CNOT6L-immunoprecipitated material (Dataset S1). Fgf21 mRNA levels were increased in Results − − Cnot6l / (Fig. 1E) and TTP-depleted hepatocytes (Fig. 2F). RIP-CHIP Identifies CNOT6L-Associated mRNAs That Contain AU-Rich − − This correlated with longer Fgf21 mRNA half-life in Cnot6l / Elements and Encode for Metabolic Regulators. The CCR4–NOT and TTP-depleted hepatocytes (T , ∼40 min) vs. wild-type and complex is a multisubunit protein machinery composed of the 1/2 control (T , ∼25 min), respectively (Fig. 3 A and B). Luciferase CCR4 (CNOT6L/CNOT6) deadenylase, the CNOT1 scaffold 1/2 reporter assays confirmed that the 3′UTR is required for CCR4– protein, and several regulatory proteins (CNOT2–CNOT11) (5), NOT/TTP-mediated degradation of Fgf21 mRNA (Fig. 3 C and has been implicated in metabolic disorders (20–23). Mam- and D). The CCR4–NOT/TTP complex also induced dead- mals have two paralogs of the CCR4 deadenylase gene, Cnot6 enylation of an Fgf21 3′UTR reporter mRNA (Fig. 3E). Cnot6l and (32, 33). CNOT6L is highly expressed in metaboli- These data demonstrate that the CCR4–NOT/TTP complex cally active tissues, such as the liver and adipose tissue, whereas promotes deadenylation and degradation of Fgf21 mRNA via CNOT6 is predominantly expressed in testis and thymus (SI ′ – interaction with its 3 UTR (Fig. 3F). Appendix, Fig. S1 A and B). To investigate the role of CCR4 Fgf21 transcription is stimulated by peroxisome proliferator- NOT in regulation of metabolism, we first identified bona fide activated receptor-α (PPAR-α) under fasting conditions, leading endogenous mRNA targets of the CCR4–NOT deadenylase in – to hepatic lipid oxidation, triglyceride clearance, and ketogenesis the liver. This was achieved by immunopurifying CCR4 NOT- (37–39). In contrast, Fgf21 mRNA is rapidly suppressed 2 h after associated mRNP complexes and analyzing their mRNA content refeeding by an unknown mechanism (37, 39). Serum FGF21 and on a transcriptome-wide scale using microarrays (Fig. 1A). He- − − hepatic Fgf21 mRNA levels were ∼2.5-fold higher under fasting / − − patocytes derived from mice lacking Cnot6l (Cnot6l ) were and refeeding conditions in Cnot6l / compared with wild-type – used as a control (SI Appendix, Fig. S1 C F). We identified 195 mice (Fig. 3 G and H). CNOT6L deficiency partially rescued CNOT6L-associated mRNAs (Dataset S1), then searched con- Fgf21 mRNA degradation by refeeding (Fig. 3I), indicating that ′ sensus sequences among 3 UTRs of 195 mRNAs (Dataset S2). reduction in serum FGF21 levels upon refeeding is mediated at These mRNAs were significantly enriched for AU-rich elements least in part by the CCR4–NOT complex. Consistently, refeeding ′ (AREs) within their 3 UTR (Fig. 1B, SI Appendix, Fig. S2A, and stimulated CNOT6L deadenylase activity in the liver (Fig. 3J and Dataset S2). AREs generally destabilize mRNAs and are found SI Appendix, Fig. S3G). These results demonstrate that the ′ within the 3 UTR of mRNAs, such as cytokines and growth CCR4–NOT/TTP complex controls hepatic FGF21 production factors that respond to acute external stimuli (34, 35). We clas- by inducing degradation of Fgf21 mRNA after feeding. sified 195 mRNAs according to their biological functions (Fig. 1C). CNOT6L-associated mRNAs exhibited enrichment Resistance to Diet-Induced Obesity, Enhanced Energy Expenditure, for those encoding metabolic factors (27% of 195 genes, P = and Improved Insulin Sensitivity in Cnot6l−/− Mice. − FGF21 pro- 2.0 × 10 3) (Fig. 1C). We selected 12 mRNAs among those motes weight and lipid reduction, and delays development of mRNAs encoding metabolism-related proteins according to the diabetes (29, 31). To examine the impact of hepatic CCR4–NOT P values and those that were associated with known metabolic on FGF21 levels in a physiological context pertinent to the devel- − − functions, and validated the interactions between CNOT6L and opment of metabolic disorders, we analyzed Cnot6l / mice, which 12 of these mRNAs using qRT-PCR (SI Appendix,Fig.S2B are viable and fertile and age without gross abnormalities (SI Ap- and C). We found that of the 12 selected mRNAs, 5 contained pendix,Fig.S4A). The mice are leaner and protected from high-fat AREs, and that the stability and steady-state levels of these diet (HFD)-induced obesity compared with wild-type mice (Fig. 4A mRNAs, such as Fgf21, Ankrd1, Socs3,andFoxk2, were in- and SI Appendix,Fig.S4A and B). Cnot6l deficiency ameliorated − − creased in Cnot6l / hepatocytes (Fig. 1 D and E and SI Ap- HFD-induced hyperglycemia, hyperlipidemia, and hyperinsulinemia pendix, Fig. S2D). These results suggest that CNOT6L decreases (Fig. 4 B–D and SI Appendix,Fig.S4C–E). Furthermore, serum

7974 | www.pnas.org/cgi/doi/10.1073/pnas.1816023116 Morita et al. Downloaded by guest on September 27, 2021 A B 30 FDR = Total RNA isolation Input arrays, wild-type 1.9 x 10-9 25 -/- Cell lysis Input arrays, Cnot6l 20 IP: anti-CNOT6L 15 IPed arrays, wild-type Wild-type or Cnot6l-/- 10 primary hepatocytes RBP IPed arrays, Cnot6l-/- with AREs (%) AAA CNOT6L 5 Percentage of mRNA Cis-acting 0 element Non-target CNOT6L-target Identification of 195 CNOT6L-associated mRNAs (Table S1)

-/- C D Wild-type Cnot6l Time after actinomycin D Time after actinomycin D Time after actinomycin D Time after actinomycin D 13% treatment (hr) treatment (hr) treatment (hr) treatment (hr) 27% 0 0.5 1 0 0.5 1 1.5 2 00.51 01234 100 100 100 100 11% * ** 7% ** 6% *** mRNA (%) mRNA 4% (%) mRNA 4% 3% Fgf21 mRNA (%) Foxk2 mRNA mRNA (%) Socs3 mRNA Ankrd1 25% 10 10 10 10 CNOT6L-associated mRNAs with AREs Metabolic process E 300 Wild-type Cnot6l-/- Cell signaling MEDICAL SCIENCES Developmental process 250 *** *** Immune system process 200 * Response to stimulus *** *** ** Transport 150 ** ** Cell cycle 100 Apoptotic process Input mRNA Others (% of wild-type) 50 0 Brpf Ctgf Pfkfb Pdk4 Ascl3 Acot4 Fgf21 Foxk2 Socs3 β -actin Slc2a1 Tnrc6a Ankrd1 mRNAs with AREs mRNAs without AREs

Fig. 1. RIP-CHIP identified CNOT6L-associated mRNAs, many of which contain AREs and encode metabolic regulators. (A) Schematic diagram of RIP-CHIP in wild-type and Cnot6l−/− primary hepatocytes. Primary hepatocytes were isolated from wild-type and Cnot6l−/− livers. Each sample was divided into two for total RNA extraction (upper arrows) and anti-CNOT6L immunoprecipitation followed by RNA extraction (lower arrows). Input and immunoprecipitated (IPed) − − RNA were hybridized to separate microarrays. Cnot6l / hepatocytes were used as a negative control. (B) Percentage of nontarget mRNAs containing AREs (blue bar) and CNOT6L-target mRNAs containing AREs (red bar). The bar graph was generated from Dataset S1.(C) Functional classification of 195 CNOT6L- − − associated mRNAs using the Panther Classification System. (D) Stability of the indicated mRNAs in wild-type and Cnot6l / hepatocytes. Hepatocytes were incubated with actinomycin D for the indicated times. Levels of the indicated mRNAs were determined by RT-qPCR, normalized to the level of Hprt mRNA, expressed as percent change of the initial mRNA level, and plotted semilogarithmically. n = 3 per group. (E) Input levels of the indicated mRNAs in wild-type − − and Cnot6l / hepatocytes were determined by RT-qPCR. n = 3 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. A representative experiment of two independent experiments (each carried out in triplicate) is presented.

− − − − levels of ketone body β-hydroxybutyrate were increased in Cnot6l / Cnot6l / liver appeared smaller (∼25%) than control (Fig. 5 A mice (Fig. 4E and SI Appendix,Fig.S4F), suggesting enhancement and B). Smaller lipid droplets and lower hepatic triglyceride − − in fatty-acid oxidation. Finally, Cnot6l deletion significantly accumulation were observed in Cnot6l / liver on an HFD improved HFD-induced glucose tolerance and alleviated insulin compared with control (Fig. 5 C and D). Moreover, Cnot6l de- resistance compared with control wild-type mice that exhibited letion led to alterations in expression of genes relevant to energy typical metabolic syndrome phenotypes (Fig. 4 F and G). We next expenditure and fat metabolism, such as Pgc-1α, Scd1, and Cd36 measured whole-body energy metabolism and found that loss of mRNAs in the liver (Fig. 5E and SI Appendix, Fig. S5B). In − − Cnot6l resulted in a significant increase in oxygen consumption agreement with the enhanced energy expenditure in Cnot6l / (Fig. 4 H and I and SI Appendix, Fig. S4 G and H). There were no mice, less fat accumulation and smaller adipocytes were ob- − − differences in food intake or locomotor activity, but increased heat served in Cnot6l / BATs (Fig. 5 F–H). Cnot6l ablation engen- production and decreased respiratory exchange ratio were ob- dered expression of Ucp1 and Pgc-1α, which are induced by served (Fig. 4 J–M and SI Appendix,Fig.S4I–L). FGF21 treatment (40–43), in BAT and subcutaneous WAT − − Cnot6l / mice on an HFD exhibited significantly reduced weight (sWAT) (Fig. 5 I and J and SI Appendix,Fig.S5C and D)(40–43). of liver, white adipose tissue (WAT), and brown adipose tissue Consequently, the weight and adipocyte size of sWAT and epi- − − (BAT) compared with wild-type mice (SI Appendix, Fig. S5A). didymal WAT (eWAT) were significantly decreased in Cnot6l / While an HFD-induced hepatic steatosis in wild-type mice, compared with control mice (Fig. 5 K–N). These results indicate

Morita et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7975 Downloaded by guest on September 27, 2021 ACPulldown B E Control TTP KD Flag-CNOT6L ++ Time after actinomycin D Time after actinomycin D treatment (hr) treatment (hr) TTP KD TTP HA-TTP - + Control TTP 00.51 012 CNOT6L 100 100 TTP CNOT6L IP: HA Input MBP-TTP-WT MBP-TTP-F319A MBP alone MBP α CNOT6L -tubulin CNOT6L * β * CNOT1 TTP Iuput -actin CNOT3 CNOT7 mRNA (%) Fgf21 mRNA MBP-TTP 10 (%) Ankrd1 mRNA 10 Time after actinomycin D Time after actinomycin D Immunoprecipitated mRNA D treatment (hr) treatment (hr) (IP: anti-CNOT6L) 00.51 01234 Control TTP KD 140 100 100 120 ** * 100 80 60 mRNA (%) Foxk2 mRNA 40 (%) Socs3 mRNA * * 10 10 20 ** CNOT6L-target with AREs ND IPed mRNA (% of control) IPed mRNA 0 Fgf21 Ankrd1 Socs3 Foxk2 Acsl3 Tnrc6a ARE targets Non-ARE targets Time after actinomycin D Time after actinomycin D treatment (hr) treatment (hr) F Input mRNA 01234 01234 Control TTP KD 250 *** 100 100 ** 200 * 150 * (%) mRNA 100 -actin β mRNA (%) Tnrc6a mRNA 10 10 50 CNOT6L-target without ARE Input mRNA (% of control) Input mRNA 0 Fgf21 Ankrd1 Socs3 Foxk2 Acsl3 Tnrc6a β-actin ARE targets Non-ARE targets

Fig. 2. TTP recruits the CCR4–NOT complex to ARE-containing target mRNAs destined for degradation. (A) Interaction of the CCR4–NOT complex with WT or mutant (F319A) TTP protein in hepatocyte extracts. Western blots of the indicated proteins from recombinant maltose binding protein (MBP)-tagged TTP proteins immobilized on amylose beads and incubated with hepatocyte extracts. (B) Interaction of Flag-CNOT6L with HA-TTP in hepatocytes examined by coimmunoprecipitation with anti-HA antibody, followed by Western blot with anti-Flag antibody. (C) Western blots of the indicated proteins in control and TTP knockdown (KD) hepatocytes. α-Tubulin and β-actin were used as loading controls. (D) Association of CNOT6L with the indicated mRNAs in the lysates from control and TTP KD hepatocytes. Immunoprecipitated (IPed) and input RNAs were isolated from anti-CNOT6L immunoprecipitates and total cell lysates, respectively. Levels of IPed and input mRNAs were determined by RT-qPCR. IPed mRNA levels were normalized to those of input mRNA. n = 3 per group. (E) Stability of the indicated mRNAs in control and TTP KD hepatocytes. Hepatocytes were incubated with actinomycin D for the indicated times. Levels of the indicated mRNAs were determined by RT-qPCR, normalized to the level of Hprt mRNA, expressed as percent change of the initial mRNA level, and plotted semilogarithmically. n = 3 per group. (F) Input levels of the indicated mRNAs in the lysates from control and TTP KD hepatocytes. n = 3 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ND, not determined; Student’s t test. For D–F, a representative experiment of two independent experiments (each carried out in triplicate) is presented.

− − that loss of CNOT6L activity dramatically improves HFD-induced weight phenotype in Cnot6l / mice is attributed by the systemic metabolic disorders. effects caused by the lack of CCR4–NOT liver activity. Restoring CNOT6L levels in the liver significantly decreased Ucp1 levels in Hepatic CNOT6L Deadenylase Activity Systemically Controls Lipid BATs and eWAT (Fig. 6 E and F), increased the size of lipid Metabolism, Steatosis, and Whole-Body Metabolism. To support droplets in adipose tissues and eWAT weight (Fig. 6 C and G), our observation that hepatic CNOT6L deadenylase activity plays and reversed serum insulin and blood glucose levels (Fig. 6 H an important role in metabolic regulation, we reintroduced a and I). In striking contrast, the E239A mutant of CNOT6L failed − − wild-type or an inactive (E239A) CNOT6L mutant (33, 44) into to reverse the phenotypes of Cnot6l / mice (Fig. 6 A–I and SI −/− Cnot6l liver by adenoviral-mediated gene transduction (SI Appendix, Fig. S6 A and B). Taken together, these data highlight Appendix, Fig. S6A) and maintained the mice on an HFD for the contribution of hepatic CNOT6L deadenylase activity in 2 wk. Liver-specific expression of wild-type CNOT6L signifi- − − metabolic disorders and whole-body energy homeostasis. cantly increased liver weight and triglyceride content in Cnot6l / mice (Fig. 6 A–C). Notably, CNOT6L partially reversed the re- FGF21 Is the Mediator of CNOT6L-Dependent Hepatic Steatosis and − − sistance of Cnot6l / mice to HFD-induced obesity (Fig. 6D and Obesity. To determine whether FGF21 mediates the effects of the SI Appendix, Fig. S6B). This demonstrates that the reduced hepatic CCR4–NOT/TTP complex on whole-body metabolism,

7976 | www.pnas.org/cgi/doi/10.1073/pnas.1816023116 Morita et al. Downloaded by guest on September 27, 2021 -/- A Wild-type Cnot6l-/- B Control TTP KD C Wild-type Cnot6l 120 *** *** 0.8 10 0.8 ** 0.7 * 0.7 8 100 8 0.6 0.6 80 mRNA (hr) mRNA 0.5 (hr) mRNA 0.5 6 6 60 0.4 0.4 4 mRNA (hr) Fgf21 mRNA 4 0.3 β -actin 0.3 40 (% of control)

0.2 0.2 Luciferase activity 2 2 20 0.1 0.1 0 0 0 0 0 (hr) Half-life of Fgf21 mRNA Half-life of Control 3'UTR of Half-life of β -actin Half-life of Fgf21 Wild-type TTP KD D E GST GST-TTP F **** ** The CCR4-NOT 120 A- 0 0.5 1 3 A- 0 0.5 1 3 Time (hr) complex 100 CNOT1 80 3’UTR-A98 TTP 60 AAn ARE 40 CNOT6L (% of control) 3’UTR-A0 3'UTR of Luciferase activity 20 Fgf21 mRNA 0 Control 3'UTR of Fgf21 G H I J Wild-type Cnot6l-/- Wild-type Cnot6l-/- Wild-type IP: anti- Cnot6l-/- CNOT6L 5.0 ** 300 300 * 4.0 250 ** 250 Fasted Re-fed

200 MEDICAL SCIENCES 3.0 200 A20 150 150 2.0 100 * 100

1.0 ** (% of wild-type) 50 50 (% of fasted wild-type) Serum FGF21 (ng/ml) mRNA in the liver Fgf21 mRNA

0 0 Remaining Fgf21 mRNA in the liver after re-feeding 0 Fasted Re-fed Fasted Re-fed

Fig. 3. The CCR4–NOT/TTP complex degrades the ARE mRNA for hepatokine FGF21 in response to feeding. (A and B) Half-lives of Fgf21 and β-actin mRNAs in − − wild-type and Cnot6l / hepatocytes (A) or in control and TTP KD hepatocytes (B). Half-lives of A and B were calculated from Figs. 1D and 2E, respectively. n = − − 3 per group. (C and D) Relative luciferase activities of luciferase construct with or without 3′UTR of Fgf21 mRNA in Cnot6l / (C) or TTP-depleted (D) he-

patocytes. n = 3 per group. (E) Deadenylation assay of Fgf21-3′UTR-A98 RNA in Krebs ascites extract in the presence of recombinant GST or GST-tagged TTP. Polyadenylated and deadenylated RNAs are marked on the right. (F) Model for structural organization of Fgf21 mRNA-bound TTP in the complex with CNOT6L deadenylase (9). (G and H) Serum FGF21 protein (G) and hepatic Fgf21 mRNA levels (H) of wild-type and Cnot6l−/− mice following 4-h refeeding after 24-h fasting. n = 8 per group. (I) Remining Fgf21 mRNA in the liver of refed wild-type and Cnot6l−/− mice was calculated from Fig. 3H. n = 8 per group. (J)

Deadenylation assay of 20-mer poly(A) RNA (A20) in anti-CNOT6L immunoprecipitates from livers of fasted or refed mice. The 20 mer poly(A) RNA is marked on the right. Validation of the immunoprecipitation is shown in SI Appendix, Fig. S3G. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test for A, B, and G–I, and two-way ANOVA with Tukey’s post hoc test for C and D.

we depleted FGF21 (SI Appendix,Fig.S6C) using adenovirus- metabolic control (SI Appendix,Fig.S6K). Thus, targeting CNOT6L delivered short-hairpin RNA (shRNA) (39, 45). Fgf21 shRNA re- could potentially provide better options for the treatment of meta- versed the beneficial effects of CNOT6L loss on liver weight, hepatic bolic disorders with fewer side effects than FGF21-based therapies. lipid droplet content, and the resistance to HFD-induced triglyceride accumulation (Fig. 6 J–L). In addition to partial res- Discussion toration of Ucp1 expression in BAT and insulin levels in serum Selective deadenylation by the CCR4–NOT complex contributes − − (SI Appendix, Fig. S6 D and E), Fgf21 knockdown in Cnot6l / significantly to the wide range of mRNA half-lives and is medi- mice resulted in increased body weight (Fig. 6M and SI Appendix, ated by specific RBPs that recruit the complex to target mRNAs, Fig. S6F). These findings further support the tenet that FGF21 is as has been described for TTP (9), Roquin (10), and miRISC (14– a major mediator of the systemic metabolic effects of CCR4– 16). RIP-CHIP analysis of CNOT6L-associated mRNAs in the NOT and that the hepatic CCR4–NOT/TTP/FGF21 axis plays an liver revealed a TTP-dependent posttranscriptional program essential role in whole-body energy homeostasis. that systemically alters mammalian metabolism. CNOT6L tar- FGF21-based therapies have shown promise for the treatment gets a subset of metabolism-related mRNAs, such as Fgf21 of metabolic disorders in humans; however, concerns have been mRNA, whose expression is rapidly altered in response to changes raised due to side effects reported in mice, including changes in in feeding conditions (37, 39, 47). In accordance with previous − − bone development and homeostasis (29, 46). Strikingly, Cnot6l / reports (9, 11, 12), the association between the CCR4–NOT com- mice did not display any defects in bone homeostasis (SI Appendix, plex and ARE mRNAs depends on TTP expression (Figs. 2 and 3). Fig. S6 G–J), suggesting that induction of FGF21 via inhibition of Together, these data demonstrate that CCR4–NOT selectively CNOT6L could circumvent bone homeostasis issues associated controls TTP-specific ARE mRNAs encoding metabolic factors in with administration of FGF21. In summary, our data establish a hepatocytes. Although the impact of TTP on immune regulation in strong link between CNOT6L deadenylase-mediated regulation mammals is well documented (36), these results ascribe a function of the hormone-like protein FGF21 and downstream systemic for TTP in organismal metabolism.

Morita et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7977 Downloaded by guest on September 27, 2021 A B Wild-type C Wild-type D Wild-type E Wild-type 50 Cnot6l-/- Cnot6l-/- Cnot6l-/- Cnot6l-/-

40 300 ** 160 * 14 * 3.5 *** ** *** 140 3.0 ********* 250 12 30 ****** ****** 120 2.5 ***** 200 10 * 100 20 8 2.0 Wild-type 150 80

Body weight (g) 1.5 10 Cnot6l-/- 6 100 60 4 1.0 0 40 * 50 0.5

8 101214161820 Serum insulin (ng/ml) 2

Blood glucose (mg/dL) 20

Age (weeks) 0 Serum triglyceride (mg/dL) 0 0 0

Serum β -hydroxybutyrate (mM) Fed Fasted HFD Fed Fasted Fed Fasted Fed Fasted HFD HFD HFD HFD F Wild-type HFD Wild-type SD G Wild-type HFD Wild-type SD H 4000 Cnot6l-/- HFD Cnot6l-/- SD Cnot6l-/- HFD Cnot6l-/- SD 400 3000 100 300 80 2000

* ** (ml/kg/hr) 60 * 2 200 * Wild-type HFD ** VO 1000 -/- ** 40 Cnot6l HFD

** blood glucose (%) 100 Percent change of 20 Dark Light 0 Blood glucose (mg/dL) 0 0 6 12 18 24 0 0306090120 0 30 60 90 120 Time (hr) Time after insulin injection (min) Time after glucose injection (min)

Wild-type Wild-type Wild-type Wild-type Wild-type I Cnot6l-/- J Cnot6l-/- K Cnot6l-/- L Cnot6l-/- M Cnot6l-/- ** ** 0.06 350 ** 0.90 3000 30 0.05 300 0.85 0.04 250 20 * 2000 200 0.03 0.80 (ml/kg/hr) RER

2 150 1000 0.02 10 VO 100 0.75 Daily food intake / 0.01 body weight (g/g/day) 50 0 0 0 Dark Light 0 0.70 Heat production (kcal/day/kg) Dark Light HFD Locomotor activity (counts/min) HFD HFD HFD HFD

Fig. 4. Protection from diet-induced metabolic disorders and enhanced energy expenditure in Cnot6l−/− mice. (A) Growth curve of wild-type and Cnot6l−/− mice fed on an HFD. HFD feeding started at 8 wk of age. n = 14–17 per group. (B–E) Levels of blood glucose (B), serum triglycerides (C), serum insulin (D), and serum β-hydroxybutyrates (E) in wild-type or Cnot6l−/− mice fed on an HFD during ad libitum feeding or fasting. n = 7–10 per group. (F and G) Intraperitoneal − − glucose tolerance tests (GTTs) (F) and insulin tolerance tests (ITTs) (G) in wild-type and Cnot6l / mice fed on an HFD or standard diet (SD). Blood glucose levels − − were measured at the indicated time points following intraperitoneal injection of glucose or insulin. n = 7–10 per group. *P < 0.05, **P < 0.01 for Cnot6l / on −/− HFD versus wild-type on HFD. (H and I) Oxygen consumption (VO2) over 24 h (H) and average VO2 (I) in wild-type and Cnot6l mice fed on HFD. VO2 were normalized to body weight. n = 5 per group. (J–M) Daily food intake per body weight (J), locomotor activity (K), calculated heat production (L), and respiratory exchange ratio (M) in wild-type and Cnot6l−/− mice fed on an HFD. n = 5 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Tukey’s post hoc test for A–G and I and Student’s t test for J–M.

Hormone-like proteins, whose expression is tightly controlled posttranscriptional regulation of Fgf21 mRNA by the CCR4– in response to nutrients, systemically control whole-body me- NOT deadenylase is critical for the repression of triglyceride tabolism (25, 26). Most studies have focused on transcriptional clearance and fatty acid oxidation following feeding. Consistent − − regulators of hormone-like proteins. For example, Fgf21 tran- with the increased FGF21 serum level in Cnot6l / mice, scription is stimulated by the transcription factor PPAR-α under CNOT6L ablation leads to an increase in serum ketone bodies, fasting conditions, leading to hepatic lipid oxidation, triglyceride oxygen consumption, and expression of genes involved in energy clearance, and ketogenesis (37–39, 47). In contrast, Fgf21 mRNA expenditure and fatty acid oxidation (Figs. 4 and 5). Thus, our is rapidly suppressed 2 h after refeeding by an unknown mech- data show that Fgf21 mRNA stability, in addition to transcrip- anism (37, 39). We show that this suppression is less pronounced tion, plays an important role in balancing serum FGF21 levels to − − in Cnot6l / liver (Fig. 3I), which demonstrates that feeding- maintain metabolic homeostasis in response to nutrients. − − induced suppression of Fgf21 mRNA is at least in part con- The obesity-resistant phenotype of Cnot6l / mice is largely, trolled by the CCR4–NOT deadenylase. Accordingly, the CCR4– but not completely, reversed by reexpression of CNOT6L in the NOT complex is activated following feeding (Fig. 3J). Thus, liver (Fig. 6), demonstrating a major role for hepatic CNOT6L in

7978 | www.pnas.org/cgi/doi/10.1073/pnas.1816023116 Morita et al. Downloaded by guest on September 27, 2021 AB-/- C D-/- Wild-type Cnot6l Liver Liver Wild-type Cnot6l Wild-type Cnot6l-/- 300 * Wild-type Cnot6l-/- * 3000

HFD 200 2000

1000 100 Liver triglyceride Liver weight (mg) μ g/mg of liver weight)

SD ( 0 0 SD HFD HFD SD HFD

E Liver F Wild-type Cnot6l-/- -/- Wild-type Cnot6l 400 *** 900 160 800 * 140 300 700 * 120 600 * 100 200 500 80 400 * * * * * 60 * * 100 300 * weight (mg) BAT 200 * 40 20 mRNA (% of wild-type) mRNA mRNA (% of wild-type) mRNA 100 0 SD HFD 0 0 G BAT Vldlr Scd1 Fabp Cd36 Acot2 Cidec Elovl6 -/- Pgc-1 α MEDICAL SCIENCES Cyp7b1 Hsd3b5 Wild-type Cnot6l Cyp2b10 Energy expenditure Lipogenesis Lipid uptake

Fat metabolism and transport HFD

H BAT I BAT J sWAT -/- Cnot6l-/- Cnot6l-/- Wild-type Cnot6l Wild-type 1200 Wild-type 300 * 1000 * HFD 800 200 ** * 600 * 400 100 * SD 200 * mRNA (% of wild-type) mRNA mRNA (% of wild-type) mRNA 0 0 β β α α α Ucp1 Ucp1 Nr4a1 Cpt1 Pgc-1 Pgc-1 Pgc-1 Pgc-1 -/- K Wild-type Cnot6l-/- L Wild-type Cnot6l-/- MNWild-type Cnot6l eWAT 2500 *** 2500 ** Wild-type Cnot6l-/-

2000 2000 HFD 1500 1500 sWAT

1000 1000 * * sWAT weight (mg) sWAT 500 weight (mg) eWAT 500 SD

0 0 eWAT SD HFD SD HFD HFD

Fig. 5. Cnot6l deletion leads to a resistance to lipid accumulation and an increase in energy expenditure in the liver, BAT, and WAT. (A–D) Weight (A), representative image (B), representative H&E staining (C), and triglyceride contents (D) of livers from wild-type and Cnot6l−/− mice fed on a SD or HFD. n = 8– 12 per group. (Scale bars, 50 μm.) Liver triglyceride contents were normalized to liver weight. (E) Expression of genes involved in energy expenditure, fatty − − acid oxidation, lipogenesis, and lipid uptake and transport in the liver of wild-type and Cnot6l / mice fed on an HFD. n = 4–6 per group. (F–H) Weight (F), − − representative image (G), and representative H&E staining (H) of BAT of wild-type and Cnot6l / mice fed on an SD or HFD. n = 8–12 per group. (I–J)Ex- − − pression of the indicated mRNAs in BAT (I) and sWAT (J) of wild-type and Cnot6l / mice fed on an HFD. n = 5–7 per group. (K–N) Weight of sWAT (K)and − − eWAT (L), representative sWAT and eWAT (M), and representative H&E staining of eWAT (N) from wild-type and Cnot6l / mice fed on SD or HFD. n = 8– 12 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test for E, I, and J and two-way ANOVA with Tukey’s post hoc test for A, D, F, K, and L. (Scale bars, H and N,50μm.)

Morita et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7979 Downloaded by guest on September 27, 2021 AB C Wild-type Cnot6l-/- 120 120 * * ** * Mock Mock CNOT6L-WT CNOT6L-E239A 100 100 80 80

60 60 Liver 40 40 (% of wild-type) 20 Liver triglyceride 20

Liver weight (% of wild-type) 0 0 BAT

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Cnot6l-/- + CNOT6L-E239A eWAT

D E F GIH 120 * * 120 120 120 ** ** 200 600 * * * * * 180 * * 100 100 160 500 100 100 80 140 400 80 80 80 120 60 100 300 60 60 60 80 40 40 60 200 40 40 (% of wild-type) (% of wild-type) 40 (% of wild-type) Gain of body weight 20 in BAT Ucp1 mRNA 100 20 20 20 20 in eWAT Ucp1 mRNA 0 0 0 0 0 0 eWAT weight (% of wild-type) eWAT Serum insulin (% of wild-type) Blood glucose (% of wild-type)

J shGFP shFGF21 K Liver L shGFP shFGF21 M shGFP shFGF21 -/- 120 * Wild-type Cnot6l 120 ** * 120 *** * *** 100 100 100

80 shGFP 80 80

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20 20 Gain of body weight 20 shFGF21 0 0 0 Wild-type Cnot6l-/- Wild-type Cnot6l-/- Wild-type Cnot6l-/-

Fig. 6. Systemic regulation of whole-body metabolism by the CCR4–NOT/FGF21 axis. (A–I) Liver weight (A), liver triglyceride contents (B), representative H&E staining of the liver, BAT, and eWAT (C), gain of body weight (D), Ucp1 mRNA level in BAT (E) and eWAT (F), eWAT weight (G), serum insulin (H), and blood − − glucose levels (I) of wild-type and Cnot6l / mice injected with adenovirus expressing EGFP, CNOT6L-WT, or CNOT6L-E239A. Ten-week-old mice were ad- ministered with adenovirus and fed on HFD for 2 wk. (Scale bars, 50 μm.) Liver triglyceride contents were normalized to liver weight. n = 7–8 per group. (J–M) − − Liver weight (J), representative liver H&E staining (K), liver triglyceride contents (L), and gain of body weight (M) of wild-type and Cnot6l / mice injected with adenovirus expressing control (shGFP) or Fgf21 shRNA (shFGF21). Ten-week-old mice were injected with adenovirus and fed on HFD for 2 wk. (Scale bars, 50 μm.) Liver triglyceride contents were normalized to liver weight. n = 10 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two- way ANOVA with Tukey’s post hoc test.

regulating systemic metabolism. The partial rescue in metabolic FGF21 is a bona fide therapeutic target that has been explored sensitivity suggests that CNOT6L activity in other metabolic in the clinic (29). Treatment with FGF21 ameliorated several tissues, such as BAT and WAT, contributes to the regulation of metabolic disorders, such as obesity, hyperlipidemia, and hy- whole-body metabolism. Furthermore, knockdown of Fgf21 in perglycemia in a variety of species, including rodents, monkeys, −/− – the liver of Cnot6l mice restores sensitivity to nutrient excess and humans (41, 42, 48 54). However, the development of and reverses the decrease seen in diet-induced weight gain (Fig. FGF21 as a drug is challenging due to its short half-life in blood = ∼ 6). It is evident that there is a significant rescue of liver-specific (T1/2 0.5 2 h), and the aggregation of its recombinant form CNOT6L function by Fgf21 knockdown by comparing the extent (55). An FGF21 analog, LY2405319, whose efficacy was validated in humans, was developed to address these issues (54). Un- of rescue by CNOT6L reexpression versus Fgf21 knockdown on fortunately, its half-life remains relatively short (T1/2 = 1.5 ∼ 3 h), body weight. These results provide compelling evidence that which motivated a search for other strategies to increase FGF21 is the major effector of CNOT6L function in the liver, FGF21 levels in vivo (29). Additionally, exogenous administra- but also indicate that CNOT6L controls some Fgf21-unrelated tion of FGF21 has been used at a concentration of 5- to 10-fold pathways relevant to systemic metabolism. Our RIP-CHIP data higher than endogenous levels (29). We found that CNOT6L show that ∼27% of mRNAs associated with CNOT6L encode ablation alleviated metabolic disorders with only a 2.5-fold in- metabolism-related factors. Characterization of these CNOT6L crease in serum FGF21 levels (Fig. 3G). These findings dem- targets should provide additional insight into metabolic control onstrate that a modest increase in basal FGF21 levels through by CCR4–NOT. mRNA stabilization is sufficient to ameliorate hepatic steatosis,

7980 | www.pnas.org/cgi/doi/10.1073/pnas.1816023116 Morita et al. Downloaded by guest on September 27, 2021 and result in more sustained effects compared with transient unpaired) when there were only two groups. All statistical analyses were administration of FGF21. Importantly, the 2.5-fold increase we performed using IBM SPSS Statistics v22 software, and the differences were observed did not cause deleterious effects on bone density, un- considered significant when P < 0.05. For detailed in vivo and in vitro ex- like exogenous FGF21 (SI Appendix, Fig. S6). Coupled with the perimental methods, see SI Appendix. observation in humans, which show a common variant in the locus of CNOT6L correlating with altered blood cholesterol ACKNOWLEDGMENTS. We thank I. Saito for the pAxCAwtit vector; M. Fisher levels (NCBI PheGenI) (56), posttranscriptional control of for providing the pAd-shFGF21 vector; J. St-Pierre, D. Pearl, C. Chapat, and S. Tahmasebi for text proofreading; A. Sylvestre, K. Kitazawa, and H. Adachi FGF21 by CNOT6L underscores the therapeutic potential of for technical assistance; the Animal Facility and the Histology Facility at the targeting CNOT6L for metabolic disorders. Goodman Cancer Research Centre for mouse work and tissue processing; and C. Lister, I. Harvey, C. Sgherri, and S. Perreault for assistance. This work was Methods supported by Canadian Institutes of Health Research Grants CIHR MOP-93607 All animal experiments were conducted according to the guidelines for (to N. Sonenberg) and MOP-125885 (to V.G.); Terry Fox Research Institute animal use issued by the Committee of Animal Experiments, McGill University, Grant TFF-116128 (to V.G., I.T., and N. Sonenberg); and the Ministry of Educa- and Institute of Medical Science, University of Tokyo. Molecular studies were tion, Culture, Sports, Science and Technology, Japan Grant-in-Aid for Scientific performed according to routine protocol previously published by our group Research 19390070 (to T.Y.) and 18K07237 (to M.M.). M.M. is supported by the (57). Differences among groups were compared using two-way ANOVA UT Rising Stars Award from the University of Texas System. O.L. is supported by followed by between-group comparison with Tukey’s post hoc test, one-way the Wallenberg Academy Fellows Program and the Swedish Research Council. ANOVA with Bonferroni’s post hoc test, or Student’s t test (two-tailed, I.T. is a Junior 2 Research Scholar of the Fonds de Recherche du Québec–Santé.

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