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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 splicing in a subset of SRSF2 target genes. (A) Splicing analysis of SRSF2 target genes by RT-PCR in FTO-depleted cells. Red boxes indicate the spliced internal exons. % exc (exclusion) indicates the exclusion level of the internal exons. The expression of Actin was detected to confirm the equal loading of PCR products. (B) Statistical analysis of the relative exclusion level of spliced exons in FTO-depleted 3T3-L1 cells to that in control cells. ***P < 0.0001, *P < 0.05. Results are shown as mean ± SD. (C) Detection of m6A levels around splicing sites of FTO target genes by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (D) Effects of FTO depletion on SRSF2 binding ability to its target genes validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. See also Supplementary information, Figure S8.
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fig5: FTO regulates splicing in a subset of SRSF2 target genes. (A) Splicing analysis of SRSF2 target genes by RT-PCR in FTO-depleted cells. Red boxes indicate the spliced internal exons. % exc (exclusion) indicates the exclusion level of the internal exons. The expression of Actin was detected to confirm the equal loading of PCR products. (B) Statistical analysis of the relative exclusion level of spliced exons in FTO-depleted 3T3-L1 cells to that in control cells. ***P < 0.0001, *P < 0.05. Results are shown as mean ± SD. (C) Detection of m6A levels around splicing sites of FTO target genes by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (D) Effects of FTO depletion on SRSF2 binding ability to its target genes validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. See also Supplementary information, Figure S8.

Mentions: To test our above hypothesis, we chose alternatively spliced genes that contain spatially overlapped SRSF2-binding sites and m6A sites around spliced exons (including 5′ and 3′ flanking exons and internal alternative exons), for alternative splicing analysis by RT-PCR. A representative gene FBXO9 showed increased m6A levels around splicing site upon FTO depletion (Supplementary information, Figure S4). We found that FTO depletion inhibited exon skipping and promoted inclusion of the alternative exons of GKAP1, ZFP809, FBXO9, KIF13A and ZFP212 genes, as shown by the decreased exclusion levels (Figure 5A, 5B and Supplementary information, Figure S6E). Supporting our findings, previously published CLIP-seq data showed that SRSF2 binds to exons around alternative splice sites of these genes45.


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 splicing in a subset of SRSF2 target genes. (A) Splicing analysis of SRSF2 target genes by RT-PCR in FTO-depleted cells. Red boxes indicate the spliced internal exons. % exc (exclusion) indicates the exclusion level of the internal exons. The expression of Actin was detected to confirm the equal loading of PCR products. (B) Statistical analysis of the relative exclusion level of spliced exons in FTO-depleted 3T3-L1 cells to that in control cells. ***P < 0.0001, *P < 0.05. Results are shown as mean ± SD. (C) Detection of m6A levels around splicing sites of FTO target genes by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (D) Effects of FTO depletion on SRSF2 binding ability to its target genes validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. See also Supplementary information, Figure S8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4260349&req=5

fig5: FTO regulates splicing in a subset of SRSF2 target genes. (A) Splicing analysis of SRSF2 target genes by RT-PCR in FTO-depleted cells. Red boxes indicate the spliced internal exons. % exc (exclusion) indicates the exclusion level of the internal exons. The expression of Actin was detected to confirm the equal loading of PCR products. (B) Statistical analysis of the relative exclusion level of spliced exons in FTO-depleted 3T3-L1 cells to that in control cells. ***P < 0.0001, *P < 0.05. Results are shown as mean ± SD. (C) Detection of m6A levels around splicing sites of FTO target genes by m6A-IP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. (D) Effects of FTO depletion on SRSF2 binding ability to its target genes validated by PAR-CLIP and RT-qPCR. ***P < 0.0001. Results are shown as mean ± SD. See also Supplementary information, Figure S8.
Mentions: To test our above hypothesis, we chose alternatively spliced genes that contain spatially overlapped SRSF2-binding sites and m6A sites around spliced exons (including 5′ and 3′ flanking exons and internal alternative exons), for alternative splicing analysis by RT-PCR. A representative gene FBXO9 showed increased m6A levels around splicing site upon FTO depletion (Supplementary information, Figure S4). We found that FTO depletion inhibited exon skipping and promoted inclusion of the alternative exons of GKAP1, ZFP809, FBXO9, KIF13A and ZFP212 genes, as shown by the decreased exclusion levels (Figure 5A, 5B and Supplementary information, Figure S6E). Supporting our findings, previously published CLIP-seq data showed that SRSF2 binds to exons around alternative splice sites of these genes45.

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