FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2206193756> ?p ?o ?g. }

• W2206193756 endingPage "2767" @default.
• W2206193756 startingPage "2756" @default.
• W2206193756 abstract "•Cnot7 and/or Tob deficiencies make mice resistant to diet-induced obesity•Expression of Cnot7 and Tob is augmented in obese iWAT and inhibits Ucp1 level•Tob interacts with BRF1 at the AU-rich region in the 3′-UTR of Ucp1 mRNA•Tob-BRF1 interaction recruits Cnot7 deadenylase to Ucp1 mRNA for its destabilization Uncoupling protein 1 (Ucp1) contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT) of mice fed a high-fat diet (HFD), Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA has an elongated poly(A) tail and persists in iWAT of Cnot7−/− and/or Tob−/− mice on a HFD. Ucp1 3′-UTR-containing mRNA is more stable in cells expressing mutant Tob that is unable to bind Cnot7 than in WT Tob-expressing cells. Tob interacts with BRF1, which binds to an AU-rich element in the Ucp1 3′-UTR. BRF1 knockdown partially restores the stability of Ucp1 3′-UTR-containing mRNA. Thus, the Cnot7-Tob-BRF1 axis inhibits Ucp1 expression and contributes to obesity. Uncoupling protein 1 (Ucp1) contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT) of mice fed a high-fat diet (HFD), Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA has an elongated poly(A) tail and persists in iWAT of Cnot7−/− and/or Tob−/− mice on a HFD. Ucp1 3′-UTR-containing mRNA is more stable in cells expressing mutant Tob that is unable to bind Cnot7 than in WT Tob-expressing cells. Tob interacts with BRF1, which binds to an AU-rich element in the Ucp1 3′-UTR. BRF1 knockdown partially restores the stability of Ucp1 3′-UTR-containing mRNA. Thus, the Cnot7-Tob-BRF1 axis inhibits Ucp1 expression and contributes to obesity. Obesity and related metabolic diseases increase the risk of diabetes, hypertension, cardiovascular diseases, and cancer. In mammals, fats accumulate in white and brown adipose tissues. White adipose tissue (WAT) stores energy in the form of triglycerides. Brown adipose tissue (BAT) dissipates stored energy as heat; thus, BAT increases energy expenditure and resistance to obesity (Harms and Seale, 2013Harms M. Seale P. Brown and beige fat: development, function and therapeutic potential.Nat. Med. 2013; 19: 1252-1263Crossref PubMed Scopus (1579) Google Scholar). Uncoupling protein 1 (Ucp1) is uniquely expressed in BAT mitochondria, where it uncouples respiration to produce heat (Rousset et al., 2004Rousset S. Alves-Guerra M.C. Mozo J. Miroux B. Cassard-Doulcier A.M. Bouillaud F. Ricquier D. The biology of mitochondrial uncoupling proteins.Diabetes. 2004; 53: S130-S135Crossref PubMed Google Scholar, Ricquier, 2011Ricquier D. Uncoupling protein 1 of brown adipocytes, the only uncoupler: a historical perspective.Front. Endocrinol. (Lausanne). 2011; 2: 85Crossref PubMed Scopus (88) Google Scholar). Ucp1 activity opposes obesity, whereas Ucp1-ablation in mice impairs thermogenesis and induces obesity (Feldmann et al., 2009Feldmann H.M. Golozoubova V. Cannon B. Nedergaard J. UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality.Cell Metab. 2009; 9: 203-209Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar). Recently, Ucp1-positive adipocytes have been also identified in white adipose deposits and like those in BAT, also dissipate stored energy (Wu et al., 2013Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (630) Google Scholar). Thermogenic programs in subcutaneous WAT that include high expression of Ucp1 protect mice from obesity (Kopecký et al., 1995Kopecký J. Clarke G. Enerbäck S. Spiegelman B. Kozak L.P. Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.J. Clin. Invest. 1995; 96: 2914-2923Crossref PubMed Scopus (486) Google Scholar, Kopecký et al., 1996Kopecký J. Hodný Z. Rossmeisl M. Syrový I. Kozak L.P. Reduction of dietary obesity in aP2-Ucp transgenic mice: physiology and adipose tissue distribution.Am. J. Physiol. 1996; 270: E768-E775PubMed Google Scholar, Seale et al., 2011Seale P. Conroe H.M. Estall J. Kajimura S. Frontini A. Ishibashi J. Cohen P. Cinti S. Spiegelman B.M. Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice.J. Clin. Invest. 2011; 121: 96-105Crossref PubMed Scopus (915) Google Scholar). Identification of regulators of Ucp1 expression will facilitate development of therapeutic approaches for treatment of obesity-related diseases. Precise regulation of gene expression is required for body homeostasis, and dysregulation of gene expression leads to various disorders, including metabolic diseases, immunological diseases, and cancer. Recently, not only transcriptional regulation, but also post-transcriptional mechanisms, including capping, splicing, and degrading of mRNAs, have attracted a great deal of attention for regulation of gene expression. Deadenylase-mediated shortening and removal of poly(A) tails at 3′-ends of eukaryotic mRNAs suppress translation and accelerate mRNA decay (Garneau et al., 2007Garneau N.L. Wilusz J. Wilusz C.J. The highways and byways of mRNA decay.Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (954) Google Scholar). The major deadenylase in mammals is the CCR4-NOT complex, which comprises at least ten subunits, Cnot1-Cnot3, Cnot6, Cnot6L, and Cnot7-Cnot11 (Albert et al., 2000Albert T.K. Lemaire M. van Berkum N.L. Gentz R. Collart M.A. Timmers H.T. Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits.Nucleic Acids Res. 2000; 28: 809-817Crossref PubMed Scopus (127) Google Scholar, Collart and Timmers, 2004Collart M.A. Timmers H.T. The eukaryotic Ccr4-not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways?.Prog. Nucleic Acid Res. Mol. Biol. 2004; 77: 289-322Crossref PubMed Scopus (107) Google Scholar). Cnot6/6L (Ccr4a/b) and Cnot7/8 (Caf1a/b), which belong to the exonuclease-endonuclease-phosphatase (EEP) family and the DEDD (Asp-Glu-Asp-Asp) family, respectively, possess deadenylase activity (Goldstrohm and Wickens, 2008Goldstrohm A.C. Wickens M. Multifunctional deadenylase complexes diversify mRNA control.Nat. Rev. Mol. Cell Biol. 2008; 9: 337-344Crossref PubMed Scopus (298) Google Scholar). Subunits of the CCR4-NOT complex are ubiquitously expressed in adult mice with some tissue preferences, such as hematopoietic and metabolic tissue (Chen et al., 2011Chen C. Ito K. Takahashi A. Wang G. Suzuki T. Nakazawa T. Yamamoto T. Yokoyama K. Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development.Biochem. Biophys. Res. Commun. 2011; 411: 360-364Crossref PubMed Scopus (32) Google Scholar, Morita et al., 2011Morita M. Oike Y. Nagashima T. Kadomatsu T. Tabata M. Suzuki T. Nakamura T. Yoshida N. Okada M. Yamamoto T. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice.EMBO J. 2011; 30: 4678-4691Crossref PubMed Scopus (63) Google Scholar). Importantly, the deadenylase activities of the CCR4-NOT complex (Morita et al., 2011Morita M. Oike Y. Nagashima T. Kadomatsu T. Tabata M. Suzuki T. Nakamura T. Yoshida N. Okada M. Yamamoto T. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice.EMBO J. 2011; 30: 4678-4691Crossref PubMed Scopus (63) Google Scholar) and Nocturnin deadenylase (Green et al., 2007Green C.B. Douris N. Kojima S. Strayer C.A. Fogerty J. Lourim D. Keller S.R. Besharse J.C. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2007; 104: 9888-9893Crossref PubMed Scopus (174) Google Scholar) are implicated in the control of obesity and energy metabolism. It appears that in liver, deadenylase activity is recruited to the 3′-end of mRNAs of energy metabolism-associated genes, shortening their lifespans and reducing their expression levels (Morita et al., 2011Morita M. Oike Y. Nagashima T. Kadomatsu T. Tabata M. Suzuki T. Nakamura T. Yoshida N. Okada M. Yamamoto T. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice.EMBO J. 2011; 30: 4678-4691Crossref PubMed Scopus (63) Google Scholar). The Tob/BTG family of antiproliferative proteins is implicated in regulation of mRNA decay (Ezzeddine et al., 2012Ezzeddine N. Chen C.Y. Shyu A.B. Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB’s deadenylation-enhancing and antiproliferative activities.Mol. Cell. Biol. 2012; 32: 1089-1098Crossref PubMed Scopus (38) Google Scholar, Ogami et al., 2014Ogami K. Hosoda N. Funakoshi Y. Hoshino S. Antiproliferative protein Tob directly regulates c-myc proto-oncogene expression through cytoplasmic polyadenylation element-binding protein CPEB.Oncogene. 2014; 33: 55-64Crossref PubMed Scopus (35) Google Scholar). Tob binds to the CCR4-NOT complex through direct interaction with Cnot7 (Horiuchi et al., 2009Horiuchi M. Takeuchi K. Noda N. Muroya N. Suzuki T. Nakamura T. Kawamura-Tsuzuku J. Takahasi K. Yamamoto T. Inagaki F. Structural basis for the antiproliferative activity of the Tob-hCaf1 complex.J. Biol. Chem. 2009; 284: 13244-13255Crossref PubMed Scopus (75) Google Scholar). Upon translation termination, Tob competes with eukaryotic release factors (eRFs) and helps recruit CCR4-NOT deadenylases to the poly(A) tails of target mRNAs, such as c-myc mRNA, via interactions with poly(A)-binding protein (PABP) and cytoplasmic polyadenylation element-binding proteins (CPEBs) (Funakoshi et al., 2007Funakoshi Y. Doi Y. Hosoda N. Uchida N. Osawa M. Shimada I. Tsujimoto M. Suzuki T. Katada T. Hoshino S. Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases.Genes Dev. 2007; 21: 3135-3148Crossref PubMed Scopus (140) Google Scholar, Ezzeddine et al., 2012Ezzeddine N. Chen C.Y. Shyu A.B. Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB’s deadenylation-enhancing and antiproliferative activities.Mol. Cell. Biol. 2012; 32: 1089-1098Crossref PubMed Scopus (38) Google Scholar, Ogami et al., 2014Ogami K. Hosoda N. Funakoshi Y. Hoshino S. Antiproliferative protein Tob directly regulates c-myc proto-oncogene expression through cytoplasmic polyadenylation element-binding protein CPEB.Oncogene. 2014; 33: 55-64Crossref PubMed Scopus (35) Google Scholar). Here, we provide evidence that deficiency of Cnot7 and Tob ameliorates diet-induced obesity. By addressing the roles of these proteins in energy metabolism, we found that stability of Ucp1 mRNA is reduced by Tob-associated Cnot7 deadenylase, which suggests involvement of post-transcriptional mechanisms in Ucp1 mRNA expression. In iWAT of mice on a high-fat diet (HFD), Ucp1 expression is decreased, and thermogenesis is attenuated, driving promotion of obesity (Kopecký et al., 1995Kopecký J. Clarke G. Enerbäck S. Spiegelman B. Kozak L.P. Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.J. Clin. Invest. 1995; 96: 2914-2923Crossref PubMed Scopus (486) Google Scholar, Kopecký et al., 1996Kopecký J. Hodný Z. Rossmeisl M. Syrový I. Kozak L.P. Reduction of dietary obesity in aP2-Ucp transgenic mice: physiology and adipose tissue distribution.Am. J. Physiol. 1996; 270: E768-E775PubMed Google Scholar, Fromme and Klingenspor, 2011Fromme T. Klingenspor M. Uncoupling protein 1 expression and high-fat diets.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2011; 300: R1-R8Crossref PubMed Scopus (138) Google Scholar, Seale et al., 2011Seale P. Conroe H.M. Estall J. Kajimura S. Frontini A. Ishibashi J. Cohen P. Cinti S. Spiegelman B.M. Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice.J. Clin. Invest. 2011; 121: 96-105Crossref PubMed Scopus (915) Google Scholar, Lee and Cowan, 2013Lee Y.K. Cowan C.A. White to brite adipocyte transition and back again.Nat. Cell Biol. 2013; 15: 568-569Crossref PubMed Scopus (17) Google Scholar). To explore the underlying mechanism of Ucp1 suppression, we assessed expression of genes encoding products that could alter thermogenesis in diet-induced obesity. Consistent with downregulation of Ucp1, expression levels of Ucp1 mRNA were dramatically decreased in iWAT of mice fed HFD, but not in mice fed a normal diet (ND), for a long term (12 weeks) (Figure 1A). In contrast, expression of other thermogenic genes (Prdm16 and Ppargc1a) and electron transport chain-regulated genes (Cox4i1, Cox5a, and Cox7a1) was similar between ND and HFD mice (Figure 1A). Because expression of Prdm16 and Ppargc1a was unchanged, we next examined expression of proteins that play a role in post-transcriptional regulation. We found that levels of Tob and subunits of the CCR4-NOT deadenylase complex, including Cnot1-3, Cnot6l, and Cnot6-8, were more than 1.5× higher in iWAT of HFD mice than in iWAT of ND mice (Figure 1B). The expression level of Cnot7 in BAT, mesenteric WAT, epididymal WAT (EpiWAT), and skeletal muscle was similar between ND and HFD mice (Figures S1A–S1D). In contrast to these observations on a long-term HFD feeding, short-term HFD feeding (4 weeks) resulted in increased expression of Ucp1 mRNA as well as transcription factors, such as Prdm16 and Ppargc1a, which positively regulate Ucp1 expression (Figure S1E). Upon short-term HFD feeding, the expression level of Tob, but not Cnot7, was also increased in iWAT (Figure S1F). After administration of a transcription inhibitor, actinomycin D, the remaining Ucp1 mRNA level was lower in iWAT of HFD-fed mice than that in ND-fed mice (Figure S1G), suggesting that Ucp1 mRNA destabilization was induced in iWAT from early stage of obesity. These results suggest that destabilization of Ucp1 mRNA correlates with increase of Tob in iWAT from early stage of HFD-induced obesity. Increased levels of CCR4-NOT subunits also contribute to suppression of Ucp1 mRNA at least in late stage of obesity. Cnot7−/− mice had significantly lower body weight than did WT mice that were fed a HFD for 12 or 24 weeks (Figures 2A and 2B ). The suppressed weight gain in Cnot7−/− mice was not associated with food intake (Figure 2C). Weights of iWAT and visceral WAT were lower in Cnot7−/− than in WT mice (Figures 2D–2F), whereas weights of spleen, thymus, kidney, heart, lung, muscle, liver, and BAT showed no significant difference between WT and Cnot7−/− mice (Figure S2A). Histological analysis using H&E staining revealed that iWAT in HFD Cnot7−/− mice contained smaller adipocytes than in HFD WT mice (Figures 2G and 2H). Circulating blood glucose levels were significantly lower in fed and fasted Cnot7−/− mice compared with WT mice (Figure 2I). Insulin and glucose tolerance tests revealed that Cnot7−/− mice had enhanced insulin sensitivity and glucose clearance (Figures 2J and 2K). ND Cnot7−/− mice had smaller iWAT masses, though not significantly, than in WT, and body weights of Cnot7−/− mice were also slightly lower than those of WT mice (Figures S2B and S2C). Taken together, Cnot7−/− deficiency reduces diet-induced iWAT adiposity and obesity in mice. To investigate the molecular mechanism underlying reduced iWAT mass in HFD Cnot7−/− mice, we performed quantitative RT-PCR analysis of genes that could regulate adipogenesis and adipocyte function. Intriguingly, we found that in the iWAT of HFD Cnot7−/− mice, Ucp1 mRNA expression was greatly increased compared with HFD WT mice. The expression enhancement reflected the length of time on HFD (>3× after 12 weeks and >12× after 24 weeks) (Figures 3A and S3A). In contrast, expression of other thermogenic genes (Prdm16, Ppargc1a, and Ucp2), electron transport chain-regulated genes (Cox4i1, Cox5a, and Cox7a1), adipogenic genes (Cebpa, Pparg1, and Pparg2), and inflammatory genes (Tnf and Ccl2) was similar between Cnot7−/− and WT mice (Figures 3A). Consistent with upregulation of Ucp1 mRNA, Ucp1 protein was also significantly more abundant in iWAT of Cnot7−/− mice (Figures 3B and 3C). In contrast, phosphorylation of hormone-sensitive lipase (HSL) at Ser660, which is relevant to lipolysis, and expression levels of Perilipin, which is a key regulator of lipid formation, were similar between HFD Cnot7−/− and WT mice (Figures 3B and 3C), suggesting that neither lipid catabolism nor anabolism was affected by the absence of Cnot7. Note that Ucp1-expressing adipocytes were increased in iWAT of Cnot7−/− mice compared with that in WT mice (Figure 3D). Ucp1 expression was significantly increased in EpiWAT and BAT of HFD Cnot7−/− mice compared with WT mice (Figures 3E and 3F), indicating that Cnot7 could inhibit Ucp1 expression in both white and BATs as well. Collectively, although Cnot7 deficiency does not affect lipolysis, adipogenesis, and inflammation, it remarkably increases Ucp1 expression in iWAT of HFD mice. Moreover, energy expenditure was augmented in HFD Cnot7−/− mice, suggesting increased oxygen consumption (Figure 3G). Finally, Ucp1 expression was not significantly altered in ND Cnot7−/− mice compared with that in ND WT mice (Figure S3B). We next examined the effect of Cnot7 on thermogenesis in adipocytes. Primary preadipocytes from iWAT of Cnot7−/− and WT mice were differentiated into mature adipocytes. Cnot7 did not affect adipogenesis per se because Cnot7−/− and WT cells underwent similar extent of differentiation, as shown by Oil-Red-O staining for lipid accumulation (Figure 4A). Cnot7−/− and WT adipocytes also expressed adipogenic genes at equivalent levels (Cebpa, Pparg1, and Pparg2) (Figure 4B). Among thermogenic genes, expression level of Ucp1, but not Prdm16, and Ppargc1a, was significantly increased in Cnot7−/− adipocytes compared with WT adipocytes (Figure 4B). Thus, Cnot7 deficiency increases Ucp1 expression in an adipose cell-autonomous manner. Note that cellular oxygen consumption in adipocytes was not altered in the presence or absence of Cnot7 (Figure S4A). We then addressed whether Cnot7 deadenylase activity is involved in regulation of Ucp1 mRNA expression. Ucp1 mRNA stability was assessed by actinomycin D-chase experiments using cultured preadipoctyes prepared from WT and Cnot7−/− mice. The half-life of Ucp1 mRNA in Cnot7−/− cells (6.7 hr) was longer than in WT cells (4.2 hr) (Figure 4C). In contrast, half-lives of Ppargc1a and Cox4i1 mRNAs were similar between Cnot7−/− and WT cells (Figure 4C). By examining the lengths of poly(A) tails, we found that Ucp1 mRNA had a longer poly(A) tail in the absence of Cnot7 than in its presence (Figure 4D). Then, we subcloned the 3′-UTR of mouse Ucp1 mRNA into the pGL3 luciferase vector. The reporter plasmid was introduced into HEK293 cells, which were subjected to actinomycin D-chase experiments in the presence of EGFP-fused human Cnot7 WT or Cnot7 H225A mutant, which lacks deadenylase activity (Horiuchi et al., 2009Horiuchi M. Takeuchi K. Noda N. Muroya N. Suzuki T. Nakamura T. Kawamura-Tsuzuku J. Takahasi K. Yamamoto T. Inagaki F. Structural basis for the antiproliferative activity of the Tob-hCaf1 complex.J. Biol. Chem. 2009; 284: 13244-13255Crossref PubMed Scopus (75) Google Scholar). The same quantities of EGFP-Cnot7 and EGFP-Cnot7 H225A proteins were expressed in HEK293 cells (Figure S4B). The half-life of luciferase-Ucp1 3′-UTR mRNA was longer in cells transfected with EGFP-Cnot7 H225A (14.7 hr) than in EGFP-Cnot7-introduced cells (7.3 hr) (Figure 4E). We further examined whether the CCR4-NOT complex interacts with Ucp1 mRNA using an RNA immmunoprecipitation assay. Because Ucp1 mRNA expression is very low in iWAT, we examined BAT lysates. In anti-Cnot3 immunoprecipitates, Cnot1, Cnot6l, and Cnot7 subunits of the CCR4-NOT complex were detected, indicating validity of the experimental system (Figure S4C). Importantly, Ucp1 mRNA was found in anti-Cnot3 immunoprecipitates (Figure 4F). Therefore, we conclude that the Cnot7 deadenylase in the CCR4-NOT complex participates in destabilization of Ucp1 mRNA. As the CCR4-NOT complex is thought to be involved in degradation of multiple mRNA species dependent on context, we further scrutinized other candidate targets. We found that the expression level of the type 2 deiodinase (Dio2) mRNA was higher in iWAT of HFD Cnot7−/− mice compared with HFD WT mice (Figure S4D). Stability of Dio2 mRNA was higher in Cnot7−/− cells than WT cells (Figure S4E). These results suggest that Cnot7 may also contribute to suppression of Dio2 mRNA in obese iWAT. Interestingly, Dio2 converts thyroxin (T4) to 3,3′,5-triiodothyronine (T3) and is involved in thermogenesis (Christoffolete et al., 2004Christoffolete M.A. Linardi C.C. de Jesus L. Ebina K.N. Carvalho S.D. Ribeiro M.O. Rabelo R. Curcio C. Martins L. Kimura E.T. Bianco A.C. Mice with targeted disruption of the Dio2 gene have cold-induced overexpression of the uncoupling protein 1 gene but fail to increase brown adipose tissue lipogenesis and adaptive thermogenesis.Diabetes. 2004; 53: 577-584Crossref PubMed Scopus (171) Google Scholar). Tob directly interacts with the Cnot7 deadenylase subunit of the CCR4-NOT complex (Horiuchi et al., 2009Horiuchi M. Takeuchi K. Noda N. Muroya N. Suzuki T. Nakamura T. Kawamura-Tsuzuku J. Takahasi K. Yamamoto T. Inagaki F. Structural basis for the antiproliferative activity of the Tob-hCaf1 complex.J. Biol. Chem. 2009; 284: 13244-13255Crossref PubMed Scopus (75) Google Scholar) and is implicated in regulation of mRNA decay (Ezzeddine et al., 2012Ezzeddine N. Chen C.Y. Shyu A.B. Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB’s deadenylation-enhancing and antiproliferative activities.Mol. Cell. Biol. 2012; 32: 1089-1098Crossref PubMed Scopus (38) Google Scholar, Ogami et al., 2014Ogami K. Hosoda N. Funakoshi Y. Hoshino S. Antiproliferative protein Tob directly regulates c-myc proto-oncogene expression through cytoplasmic polyadenylation element-binding protein CPEB.Oncogene. 2014; 33: 55-64Crossref PubMed Scopus (35) Google Scholar). As expression of both Tob and Cnot7 in iWAT of HFD mice was inversely correlated with that of Ucp1 (Figures 1A and 1B), we hypothesized that Tob is also involved in regulation of Ucp1 mRNA persistence. We first analyzed metabolic differences between HFD WT and Tob−/− mice and found that mice and their iWAT weighed less in the absence of Tob than in its presence (Figures 5A and 5B ). BAT mass was also significantly lower in Tob−/− mice than in WT mice, whereas no significant difference was detected between Tob−/− and WT mice in spleen, kidney, heart, muscle, and liver (Figure S5A). Moreover, Ucp1 mRNA expression levels were higher in iWAT of Tob−/− mice compared with WT mice (Figure 5C). Ucp1-expressing adipocytes were increased in iWAT of Tob−/− mice compared with WT mice (Figure 5D). These results suggest that Tob inhibits Ucp1 expression in iWAT. To determine whether Tob is involved in regulation of Ucp1 mRNA decay, we analyzed lengths of poly(A) tails and half-lives of Ucp1 mRNA. Poly(A)-tails were apparently longer in iWAT of Tob−/− mice than in WT mice (Figure 5E). Stability of luciferase-Ucp1 3′-UTR reporter mRNA was lower in WT Tob expressing than W93ATob-expressing HEK293 cells (Figure 5F). Because Tob W93 is important for Tob-Cnot7 interaction (Figure 5G), the data suggest that Tob together with Cnot7 participates in deadenylation-induced Ucp1 mRNA degradation. Furthermore, an RNA immunoprecipitation assay using lysates of luciferase-Ucp1 3′-UTR-expressing cells revealed that Tob interacted with luciferase-Ucp1 3′-UTR mRNA (Figure 5H) and with subunits of the CCR4-NOT complex, including Cnot1, Cnot6l, and Cnot9 (Figure S5B). Finally, flag-tagged Ucp1 3′-UTR sequences (Adachi et al., 2014Adachi S. Homoto M. Tanaka R. Hioki Y. Murakami H. Suga H. Matsumoto M. Nakayama K.I. Hatta T. Iemura S. Natsume T. ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.Nucleic Acids Res. 2014; 42: 10037-10049Crossref PubMed Scopus (50) Google Scholar) interacted, with low efficiency, with Cnot1 of the CCR4-NOT complex in HEK293 cells that overexpress Tob. The interaction barely occurred in those expressing a Tob W93A mutant (Figure 5I). The results suggest that Tob recruits Cnot7 of the CCR4-NOT complex to the 3′-UTR of Ucp1 mRNA and facilitates its deadenylation. Note that Cnot7 and Tob deficiencies did not affect expression levels of Tob and Cnot7, respectively (Figures S5C and S5D). Since the 3′-UTR sequence of Ucp1 mRNA contains AU-rich elements (AREs), including UAUUUAU (Figure 6A), we performed immunoprecipitation experiments using flag-tagged Ucp1 3′-UTR to search for RNA-binding proteins (RBPs) that interact with this element. We identified the TTP family of ARE-recognizing protein, BRF1, but not KSRP (another type of ARE-binding protein) nor TIA-1/TIAR, in immunoprecipitates (Figure 6B). To identify BRF1-interacting sequences in the Ucp1 3′-UTR, we performed a sequence-specific competition experiment using 12 bp of LNA (Locked Nucleic Acid)-oligonucleotides. The results indicated that BRF1 interacted with the Ucp1 3′-UTR through the UAUUUAU (oligo-2) sequence, but not UAUUUAC (oligo-1) nor UAUUUAA (oligo-3) sequence (Figure 6C). Other members of the TTP family of proteins, TTP and BRF2, also bound to the 3′-UTR of Ucp1 mRNA through UAUUUAU sequence (Figures S6A–S6C). Additionally, BRF1 interacted with a 3′-UTR fragment of human UCP1 mRNA, which includes a UAUUUAU sequence that localizes within the nucleotide sequence 345-414 (Figure S6D). Consistent with a previous report (Adachi et al., 2014Adachi S. Homoto M. Tanaka R. Hioki Y. Murakami H. Suga H. Matsumoto M. Nakayama K.I. Hatta T. Iemura S. Natsume T. ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.Nucleic Acids Res. 2014; 42: 10037-10049Crossref PubMed Scopus (50) Google Scholar), BRF1 interacted with the Cnot1 and Cnot7 subunits of the CCR4-NOT complex (Figure 6D). Co-immunoprecipitation experiments using lysates of flag-tagged Tob expressing cells showed that Tob interacted with BRF1 through both amino (N)- and carboxyl (C)-terminal regions (Figures 6E and S6E). Cnot1 of the CCR4-NOT complex was recruited to the Ucp1 3′-UTR by overexpression of BRF1 in HEK293 cells (Figure 6F). Moreover, stability of luciferase-Ucp1 3′-UTR reporter mRNA was higher in BRF1 siRNA-treated HEK293 cells than in control cells (Figures 6G and S6F). Finally, to determine whether the BRF1-interacting ARE in Ucp1 3′-UTR affects its mRNA stability, we generated an ARE mutant of the Ucp1 3′-UTR reporter, in which the nucleotide sequence UAUUUAU was converted to UCUUUCU. BRF1 interaction with Ucp1 3′-UTR was attenuated by the mutation (Figure 6H), and the stability of luciferase-Ucp1 3′-UTR reporter mRNA with the mutated UCUUUCU sequence was higher than that with non-mutated sequence (Figure 6I). Taken together, these results suggest that BRF1, at least in part, reduces stability of Ucp1 3′-UTR through recruitment of Tob and Cnot7 in the CCR4-NOT complex to the ARE in the Ucp1 3′-UTR. The CCR4-NOT deadenylase complex is involved in regulation of obesity. For example, deficiency of the Cnot3 subunit in mice increases expression of genes related to ATP production in the liver (Morita et al., 2011Morita M. Oike Y. Nagashima T. Kadomatsu T. Tabata M. Suzuki T. Nakamura T. Yoshida N. Okada M. Yamamoto T. Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice.EMBO J. 2011; 30: 4678-4691Crossref PubMed Scopus (63) Google Scholar). Here, we report that the Cnot7 deadenylase subunit of the CCR4-NOT complex is involved in control of obesity and adipose function. Increased expression of Cnot7 and its interacting partner Tob is observed in iWAT and correlates with accumulation of fats in iWAT. Mice deficient in Cnot7 and/or Tob store less fat than do WT mice. Accordingly, these mice are resistant to HFD-induced obesity and express remarkably increased levels of Ucp1. These observations are consistent with the observation that adipose tissue-specific Ucp1 transgenic mice show resistance to diet-induced obesity and reduction of iWAT (Kopecký et al., 1995Kopecký J. Clarke G. Enerbäck S. Spiegelman B. Kozak L.P. Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.J. Clin. Invest. 1995; 96: 2914-2923Crossref PubMed Scopus (486) Google Scholar, Kopecký et al., 1996Kopecký J. Hodný Z. Rossmeisl M. Syrový I. Kozak L.P. Reduction of dietary obesity in aP2-Ucp transgenic mice: physiology and adipose tissue distribution.Am. J. Physiol. 1996; 270: E768-E775PubMed Google Scholar). Ucp1 expression also increases in cultured Cnot7−/− adipocytes, indicating that Cnot7 suppresses Ucp1 expression in an adipose cell-autonomous manner. Oxygen consumption is regulated by multiple factors and is unchanged in Cnot7−/− adipocytes in which Ucp1 increase is selective. In support, Ucp1 overexpression in 3T3-L1 adipocytes affects oxygen consumption minimally (Si et al., 2007Si Y. Palani S. Jayaraman A. Lee K. Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism.J." @default.
• W2206193756 created "2016-06-24" @default.
• W2206193756 creator A5007643254 @default.
• W2206193756 creator A5024136589 @default.
• W2206193756 creator A5038487642 @default.
• W2206193756 creator A5039143681 @default.
• W2206193756 creator A5040045427 @default.
• W2206193756 creator A5063107400 @default.
• W2206193756 creator A5082046242 @default.
• W2206193756 date "2015-12-01" @default.
• W2206193756 modified "2023-10-12" @default.
• W2206193756 title "Post-transcriptional Stabilization of Ucp1 mRNA Protects Mice from Diet-Induced Obesity" @default.
• W2206193756 cites W1487579783 @default.
• W2206193756 cites W1824908845 @default.
• W2206193756 cites W1971284016 @default.
• W2206193756 cites W1974418613 @default.
• W2206193756 cites W1980337975 @default.
• W2206193756 cites W1987735654 @default.
• W2206193756 cites W1995100342 @default.
• W2206193756 cites W1995253518 @default.
• W2206193756 cites W2008632158 @default.
• W2206193756 cites W2008984486 @default.
• W2206193756 cites W2019768782 @default.
• W2206193756 cites W2049491436 @default.
• W2206193756 cites W2052991419 @default.
• W2206193756 cites W2058220225 @default.
• W2206193756 cites W2061034039 @default.
• W2206193756 cites W2067475896 @default.
• W2206193756 cites W2068291843 @default.
• W2206193756 cites W2076936508 @default.
• W2206193756 cites W2079090155 @default.
• W2206193756 cites W2080098347 @default.
• W2206193756 cites W2088860412 @default.
• W2206193756 cites W2102606027 @default.
• W2206193756 cites W2104645520 @default.
• W2206193756 cites W2108745351 @default.
• W2206193756 cites W2114821999 @default.
• W2206193756 cites W2119649463 @default.
• W2206193756 cites W2141138727 @default.
• W2206193756 cites W2145142659 @default.
• W2206193756 cites W2145853549 @default.
• W2206193756 cites W2151064573 @default.
• W2206193756 cites W2153101998 @default.
• W2206193756 cites W2155560632 @default.
• W2206193756 cites W2160677035 @default.
• W2206193756 cites W2160931313 @default.
• W2206193756 cites W2170744835 @default.
• W2206193756 cites W2170874580 @default.
• W2206193756 doi "https://doi.org/10.1016/j.celrep.2015.11.056" @default.
• W2206193756 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26711342" @default.
• W2206193756 hasPublicationYear "2015" @default.
• W2206193756 type Work @default.
• W2206193756 sameAs 2206193756 @default.
• W2206193756 citedByCount "46" @default.
• W2206193756 countsByYear W22061937562016 @default.
• W2206193756 countsByYear W22061937562017 @default.
• W2206193756 countsByYear W22061937562018 @default.
• W2206193756 countsByYear W22061937562019 @default.
• W2206193756 countsByYear W22061937562020 @default.
• W2206193756 countsByYear W22061937562021 @default.
• W2206193756 countsByYear W22061937562022 @default.
• W2206193756 countsByYear W22061937562023 @default.
• W2206193756 crossrefType "journal-article" @default.
• W2206193756 hasAuthorship W2206193756A5007643254 @default.
• W2206193756 hasAuthorship W2206193756A5024136589 @default.
• W2206193756 hasAuthorship W2206193756A5038487642 @default.
• W2206193756 hasAuthorship W2206193756A5039143681 @default.
• W2206193756 hasAuthorship W2206193756A5040045427 @default.
• W2206193756 hasAuthorship W2206193756A5063107400 @default.
• W2206193756 hasAuthorship W2206193756A5082046242 @default.
• W2206193756 hasBestOaLocation W22061937561 @default.
• W2206193756 hasConcept C104317684 @default.
• W2206193756 hasConcept C105580179 @default.
• W2206193756 hasConcept C126322002 @default.
• W2206193756 hasConcept C134018914 @default.
• W2206193756 hasConcept C143065580 @default.
• W2206193756 hasConcept C143128242 @default.
• W2206193756 hasConcept C151279926 @default.
• W2206193756 hasConcept C151955695 @default.
• W2206193756 hasConcept C171089720 @default.
• W2206193756 hasConcept C173396325 @default.
• W2206193756 hasConcept C185592680 @default.
• W2206193756 hasConcept C22667442 @default.
• W2206193756 hasConcept C55493867 @default.
• W2206193756 hasConcept C71924100 @default.
• W2206193756 hasConcept C81979416 @default.
• W2206193756 hasConcept C86803240 @default.
• W2206193756 hasConcept C89604277 @default.
• W2206193756 hasConcept C95444343 @default.
• W2206193756 hasConceptScore W2206193756C104317684 @default.
• W2206193756 hasConceptScore W2206193756C105580179 @default.
• W2206193756 hasConceptScore W2206193756C126322002 @default.
• W2206193756 hasConceptScore W2206193756C134018914 @default.
• W2206193756 hasConceptScore W2206193756C143065580 @default.
• W2206193756 hasConceptScore W2206193756C143128242 @default.
• W2206193756 hasConceptScore W2206193756C151279926 @default.
• W2206193756 hasConceptScore W2206193756C151955695 @default.
• W2206193756 hasConceptScore W2206193756C171089720 @default.

• next