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FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.

Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, Hao YJ, Ping XL, Chen YS, Wang WJ, Jin KX, Wang X, Huang CM, Fu Y, Ge XM, Song SH, Jeong HS, Yanagisawa H, Niu Y, Jia GF, Wu W, Tong WM, Okamoto A, He C, Rendtlew Danielsen JM, Wang XJ, Yang YG - Cell Res. (2014)

Bottom Line: The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown.Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons.These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Acaemy of Sciences, No. 1-7 Beichen West Road, Chaoyang District, Beijing 100101, China [2] University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China.

ABSTRACT
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

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FTO depletion interferes with adipogenesis. (A-C) 3T3-L1 pre-adipocytes (Day −2) were treated with FTO, ALKBH5, METTL3 or control siRNA. Forty-eight hours later, cells were lysed and subjected to immunoblotting with the indicated antibodies (A). Forty-eight hours following siRNA treatment differentiation was induced by incubation with the differentiation cocktail (IBMX/DEX/Insulin) on Day 0. Differentiation status was determined by Oil Red O staining (B) and triglyceride assay (C) on Day 0 and 10. (C) Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. Results are shown as mean ± SD. (D) 3T3-L1 cells collected at different time points (D0/2/5/10) during adipogenesis were lysed and subjected to immunoblotting with the indicated antibodies. β-tublin was used as loading control. (E) RT-PCR detected the expression levels of FTO, METTL3, as well as adipogenic markers, including ADIPSIN and PREF-1, during adipocyte differentiation. β-Actin was used as loading control. (F) mRNA was isolated from multiple stages (D0/5/10) of adipogenesis and used in dot blot analyses with m6A antibody. mRNA was loaded by two-fold serial dilution. The m6A contents are shown in the upper panel. Equal loading of mRNA was verified by methylene blue staining (lower panel). See also Supplementary information, Figure S1.
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fig1: FTO depletion interferes with adipogenesis. (A-C) 3T3-L1 pre-adipocytes (Day −2) were treated with FTO, ALKBH5, METTL3 or control siRNA. Forty-eight hours later, cells were lysed and subjected to immunoblotting with the indicated antibodies (A). Forty-eight hours following siRNA treatment differentiation was induced by incubation with the differentiation cocktail (IBMX/DEX/Insulin) on Day 0. Differentiation status was determined by Oil Red O staining (B) and triglyceride assay (C) on Day 0 and 10. (C) Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. Results are shown as mean ± SD. (D) 3T3-L1 cells collected at different time points (D0/2/5/10) during adipogenesis were lysed and subjected to immunoblotting with the indicated antibodies. β-tublin was used as loading control. (E) RT-PCR detected the expression levels of FTO, METTL3, as well as adipogenic markers, including ADIPSIN and PREF-1, during adipocyte differentiation. β-Actin was used as loading control. (F) mRNA was isolated from multiple stages (D0/5/10) of adipogenesis and used in dot blot analyses with m6A antibody. mRNA was loaded by two-fold serial dilution. The m6A contents are shown in the upper panel. Equal loading of mRNA was verified by methylene blue staining (lower panel). See also Supplementary information, Figure S1.

Mentions: To investigate the function of m6A modification in adipogenesis, we took advantage of the well-characterized mouse 3T3-L1 pre-adipocyte cell line (Supplementary information, Figure S1A). FTO, the other m6A demethylase ALKBH5 or methyltransferase METTL3 was depleted individually by siRNA and then the pre-adipocytes were induced to differentiate (Figure 1A). Interestingly, FTO depletion severely impaired differentiation as demonstrated by Oil Red O staining (Figure 1B) and triglyceride content measurements (Figure 1C). This is consistent with decreased fat tissue accumulation observed in FTO- mouse5,8,35. While METTL3 deficiency appeared to promote pre-adipocyte differentiation (Figure 1A-1C), ALKBH5 which is important for spermatogenesis34, had no obvious effect on adipogenesis (Figure 1A-1C). Since FTO and METTL3 have opposing catalytic activities, their different effects on differentiation may be caused by changes in m6A dynamics when either of them is depleted.


FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.

Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, Hao YJ, Ping XL, Chen YS, Wang WJ, Jin KX, Wang X, Huang CM, Fu Y, Ge XM, Song SH, Jeong HS, Yanagisawa H, Niu Y, Jia GF, Wu W, Tong WM, Okamoto A, He C, Rendtlew Danielsen JM, Wang XJ, Yang YG - Cell Res. (2014)

FTO depletion interferes with adipogenesis. (A-C) 3T3-L1 pre-adipocytes (Day −2) were treated with FTO, ALKBH5, METTL3 or control siRNA. Forty-eight hours later, cells were lysed and subjected to immunoblotting with the indicated antibodies (A). Forty-eight hours following siRNA treatment differentiation was induced by incubation with the differentiation cocktail (IBMX/DEX/Insulin) on Day 0. Differentiation status was determined by Oil Red O staining (B) and triglyceride assay (C) on Day 0 and 10. (C) Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. Results are shown as mean ± SD. (D) 3T3-L1 cells collected at different time points (D0/2/5/10) during adipogenesis were lysed and subjected to immunoblotting with the indicated antibodies. β-tublin was used as loading control. (E) RT-PCR detected the expression levels of FTO, METTL3, as well as adipogenic markers, including ADIPSIN and PREF-1, during adipocyte differentiation. β-Actin was used as loading control. (F) mRNA was isolated from multiple stages (D0/5/10) of adipogenesis and used in dot blot analyses with m6A antibody. mRNA was loaded by two-fold serial dilution. The m6A contents are shown in the upper panel. Equal loading of mRNA was verified by methylene blue staining (lower panel). See also Supplementary information, Figure S1.
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fig1: FTO depletion interferes with adipogenesis. (A-C) 3T3-L1 pre-adipocytes (Day −2) were treated with FTO, ALKBH5, METTL3 or control siRNA. Forty-eight hours later, cells were lysed and subjected to immunoblotting with the indicated antibodies (A). Forty-eight hours following siRNA treatment differentiation was induced by incubation with the differentiation cocktail (IBMX/DEX/Insulin) on Day 0. Differentiation status was determined by Oil Red O staining (B) and triglyceride assay (C) on Day 0 and 10. (C) Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. Results are shown as mean ± SD. (D) 3T3-L1 cells collected at different time points (D0/2/5/10) during adipogenesis were lysed and subjected to immunoblotting with the indicated antibodies. β-tublin was used as loading control. (E) RT-PCR detected the expression levels of FTO, METTL3, as well as adipogenic markers, including ADIPSIN and PREF-1, during adipocyte differentiation. β-Actin was used as loading control. (F) mRNA was isolated from multiple stages (D0/5/10) of adipogenesis and used in dot blot analyses with m6A antibody. mRNA was loaded by two-fold serial dilution. The m6A contents are shown in the upper panel. Equal loading of mRNA was verified by methylene blue staining (lower panel). See also Supplementary information, Figure S1.
Mentions: To investigate the function of m6A modification in adipogenesis, we took advantage of the well-characterized mouse 3T3-L1 pre-adipocyte cell line (Supplementary information, Figure S1A). FTO, the other m6A demethylase ALKBH5 or methyltransferase METTL3 was depleted individually by siRNA and then the pre-adipocytes were induced to differentiate (Figure 1A). Interestingly, FTO depletion severely impaired differentiation as demonstrated by Oil Red O staining (Figure 1B) and triglyceride content measurements (Figure 1C). This is consistent with decreased fat tissue accumulation observed in FTO- mouse5,8,35. While METTL3 deficiency appeared to promote pre-adipocyte differentiation (Figure 1A-1C), ALKBH5 which is important for spermatogenesis34, had no obvious effect on adipogenesis (Figure 1A-1C). Since FTO and METTL3 have opposing catalytic activities, their different effects on differentiation may be caused by changes in m6A dynamics when either of them is depleted.

Bottom Line: The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown.Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons.These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Acaemy of Sciences, No. 1-7 Beichen West Road, Chaoyang District, Beijing 100101, China [2] University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China.

ABSTRACT
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

Show MeSH
Related in: MedlinePlus