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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 regulates alternative splicing of adipogenensis-related factor RUNX1T1. (A) Alternative splicing of RUNX1T1 was identified by RT-PCR using the indicated PCR primer set (arrows). Depending on isoform, bands of either 496 bp or 245 bp can be detected. (B) Statistical analysis of the relative exclusion level of RUNX1T1 in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (C) RUNX1T1 detection in 3T3-L1 pre-adipocyte lysates shows the endogenous protein products of the two RUNX1T1 isoforms in control and FTO-deficient 3T3-L1 pre-adipocytes. An unspecific band was labeled by *. (D) Statistical analysis of RUNX1T1-S protein expression levels in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (E) Detection of m6A levels around splicing sites of RUNX1T1 gene by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (F) Effect of FTO depletion on SRSF2 binding ability to exons around splicing sites validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (G-I) 3T3-L1 pre-adipocytes were transfected with pEGFP-C1b, pEGFP-C1b-Runx1T1-L or pEGFP-C1b-Runx1T1-S. Fory-eight hours later, transfection cells were lysed and immunoblotted with the indicated antibodies (G). An unspecific band or degradation band was labeled by *. Transfected cells were induced to differentiation and subjected to triglyceride analysis (H) and Oil Red O staining (I) on D10. Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. **P < 0.01. Results are shown as mean ± SD. See also Supplementary information, Figure S9.
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fig6: FTO regulates alternative splicing of adipogenensis-related factor RUNX1T1. (A) Alternative splicing of RUNX1T1 was identified by RT-PCR using the indicated PCR primer set (arrows). Depending on isoform, bands of either 496 bp or 245 bp can be detected. (B) Statistical analysis of the relative exclusion level of RUNX1T1 in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (C) RUNX1T1 detection in 3T3-L1 pre-adipocyte lysates shows the endogenous protein products of the two RUNX1T1 isoforms in control and FTO-deficient 3T3-L1 pre-adipocytes. An unspecific band was labeled by *. (D) Statistical analysis of RUNX1T1-S protein expression levels in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (E) Detection of m6A levels around splicing sites of RUNX1T1 gene by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (F) Effect of FTO depletion on SRSF2 binding ability to exons around splicing sites validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (G-I) 3T3-L1 pre-adipocytes were transfected with pEGFP-C1b, pEGFP-C1b-Runx1T1-L or pEGFP-C1b-Runx1T1-S. Fory-eight hours later, transfection cells were lysed and immunoblotted with the indicated antibodies (G). An unspecific band or degradation band was labeled by *. Transfected cells were induced to differentiation and subjected to triglyceride analysis (H) and Oil Red O staining (I) on D10. Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. **P < 0.01. Results are shown as mean ± SD. See also Supplementary information, Figure S9.

Mentions: To validate the biological relevance of m6A-dependent regulation of SRSF2 binding and mRNA splicing, we chose to thoroughly examine the expression and function of an adipogenesis-related transcription factor, Runt-related transcription factor 1 (RUNX1T1)52. RUNX1T1 can be expressed in two isoforms. The PCR product of the constitutive isoform RUNX1T1-L is 496 bp, while that of the exon 6-skipped isoform RUNX1T1-S is 245 bp (Figure 6A). The PCR products were verified by sequencing analysis (Supplementary information, Figure S9A). In FTO-depleted 3T3-L1 cells, the exclusion level of exon 6 was decreased (Figure 6A and 6B). To the contrary, the exclusion level was increased in METTL3-depleted 3T3-L1 cells (Supplementary information, Figure S9B and S9C).


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 regulates alternative splicing of adipogenensis-related factor RUNX1T1. (A) Alternative splicing of RUNX1T1 was identified by RT-PCR using the indicated PCR primer set (arrows). Depending on isoform, bands of either 496 bp or 245 bp can be detected. (B) Statistical analysis of the relative exclusion level of RUNX1T1 in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (C) RUNX1T1 detection in 3T3-L1 pre-adipocyte lysates shows the endogenous protein products of the two RUNX1T1 isoforms in control and FTO-deficient 3T3-L1 pre-adipocytes. An unspecific band was labeled by *. (D) Statistical analysis of RUNX1T1-S protein expression levels in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (E) Detection of m6A levels around splicing sites of RUNX1T1 gene by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (F) Effect of FTO depletion on SRSF2 binding ability to exons around splicing sites validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (G-I) 3T3-L1 pre-adipocytes were transfected with pEGFP-C1b, pEGFP-C1b-Runx1T1-L or pEGFP-C1b-Runx1T1-S. Fory-eight hours later, transfection cells were lysed and immunoblotted with the indicated antibodies (G). An unspecific band or degradation band was labeled by *. Transfected cells were induced to differentiation and subjected to triglyceride analysis (H) and Oil Red O staining (I) on D10. Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. **P < 0.01. Results are shown as mean ± SD. See also Supplementary information, Figure S9.
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fig6: FTO regulates alternative splicing of adipogenensis-related factor RUNX1T1. (A) Alternative splicing of RUNX1T1 was identified by RT-PCR using the indicated PCR primer set (arrows). Depending on isoform, bands of either 496 bp or 245 bp can be detected. (B) Statistical analysis of the relative exclusion level of RUNX1T1 in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (C) RUNX1T1 detection in 3T3-L1 pre-adipocyte lysates shows the endogenous protein products of the two RUNX1T1 isoforms in control and FTO-deficient 3T3-L1 pre-adipocytes. An unspecific band was labeled by *. (D) Statistical analysis of RUNX1T1-S protein expression levels in FTO-depleted 3T3-L1 cells to that in control cells. **P < 0.01. Results are shown as mean ± SD. (E) Detection of m6A levels around splicing sites of RUNX1T1 gene by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (F) Effect of FTO depletion on SRSF2 binding ability to exons around splicing sites validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (G-I) 3T3-L1 pre-adipocytes were transfected with pEGFP-C1b, pEGFP-C1b-Runx1T1-L or pEGFP-C1b-Runx1T1-S. Fory-eight hours later, transfection cells were lysed and immunoblotted with the indicated antibodies (G). An unspecific band or degradation band was labeled by *. Transfected cells were induced to differentiation and subjected to triglyceride analysis (H) and Oil Red O staining (I) on D10. Triglyceride content was quantified and normalized to protein content. *P < 0.05 is considered significant. **P < 0.01. Results are shown as mean ± SD. See also Supplementary information, Figure S9.
Mentions: To validate the biological relevance of m6A-dependent regulation of SRSF2 binding and mRNA splicing, we chose to thoroughly examine the expression and function of an adipogenesis-related transcription factor, Runt-related transcription factor 1 (RUNX1T1)52. RUNX1T1 can be expressed in two isoforms. The PCR product of the constitutive isoform RUNX1T1-L is 496 bp, while that of the exon 6-skipped isoform RUNX1T1-S is 245 bp (Figure 6A). The PCR products were verified by sequencing analysis (Supplementary information, Figure S9A). In FTO-depleted 3T3-L1 cells, the exclusion level of exon 6 was decreased (Figure 6A and 6B). To the contrary, the exclusion level was increased in METTL3-depleted 3T3-L1 cells (Supplementary information, Figure S9B and S9C).

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