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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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m6A modification influences RNA binding ability of SRSF2. (A) Density plots showing the relative distance (X-axis), calculated by BEDTools' closestBed, between m6A sites and SRSF1-4-binding sites. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (B) The distance between the top-ranking SRSF1 and 2 ESEs predicted by ESEfinder and the nearest RRACH in identified m6A peak (red color) were calculated by BEDTools' closestBed (X axis). The distances of m6A-modified RRACH sites to the ESEs predicted by ESEfinder were calculated and their relative density distributions were shown as density plot. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (C) Analysis of the distribution of SRSF1-4-binding clusters along 5′ exon-intron and 3′ intron-exon boundaries (100 nt upstream and 300 nt downstream of 5′SS; 300 nt upstream and 100 nt downstream of 3′SS). (D) PAR-CLIP of SRSF2 in control and FTO-depleted 3T3-L1 cells transfected with the pCS2-Flag-SRSF2 plasmid. SRSF2 protein-RNA complex was pulled down with Flag M2 Affinity Gel (mouse), and then RNA was labeled and detected following instruction of biotin labeling kit. The expression levels of SRSF2 protein in whole cell lysate (WCE) was detected by western blotting with Flag antibody (rabbit). Knockdown efficiency of FTO was detected with its specific antibody. β-tubulin was used as loading control. (E) RNA binding ability of SRSF2 was calculated by normalizing binding RNAs to the corresponding pull-downed proteins. Statistical analysis shows the relative binding ability of SRSF2 in FTO-depleted cells (P < 0.0001) to that in control cells (siCTRL). See also Supplementary information, Figures S6-S7.
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fig4: m6A modification influences RNA binding ability of SRSF2. (A) Density plots showing the relative distance (X-axis), calculated by BEDTools' closestBed, between m6A sites and SRSF1-4-binding sites. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (B) The distance between the top-ranking SRSF1 and 2 ESEs predicted by ESEfinder and the nearest RRACH in identified m6A peak (red color) were calculated by BEDTools' closestBed (X axis). The distances of m6A-modified RRACH sites to the ESEs predicted by ESEfinder were calculated and their relative density distributions were shown as density plot. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (C) Analysis of the distribution of SRSF1-4-binding clusters along 5′ exon-intron and 3′ intron-exon boundaries (100 nt upstream and 300 nt downstream of 5′SS; 300 nt upstream and 100 nt downstream of 3′SS). (D) PAR-CLIP of SRSF2 in control and FTO-depleted 3T3-L1 cells transfected with the pCS2-Flag-SRSF2 plasmid. SRSF2 protein-RNA complex was pulled down with Flag M2 Affinity Gel (mouse), and then RNA was labeled and detected following instruction of biotin labeling kit. The expression levels of SRSF2 protein in whole cell lysate (WCE) was detected by western blotting with Flag antibody (rabbit). Knockdown efficiency of FTO was detected with its specific antibody. β-tubulin was used as loading control. (E) RNA binding ability of SRSF2 was calculated by normalizing binding RNAs to the corresponding pull-downed proteins. Statistical analysis shows the relative binding ability of SRSF2 in FTO-depleted cells (P < 0.0001) to that in control cells (siCTRL). See also Supplementary information, Figures S6-S7.

Mentions: SR proteins are important for splice site recognition and intron processing. They recognize the pre-mRNA cis-acting elements, ESEs, and thereby participate in the selection of splice sites. ESEs recognized by SRSF1 and 2, like m6A, are highly enriched at the edges of exons44, suggesting a potential cooperative role between SRSF1/2 and m6A in RNA splicing. To test this, we calculated the distances between SRSF1-4-binding clusters based on the previously published CLIP-seq datasets45,46 and the nearest m6A sites. As a control, randomly selected sites with the same length as the m6A peak within the same exons were generated by BEDTools' shuffleBed. There was a significant spatial overlap between SRSF1- and 2-binding clusters and m6A sites (Figure 4A). In contrast, no overlap between SRSF3, 4 or ribonucleoproteins hnRNPC/hnRNRH47,48 and m6A sites was detected (Figure 4A and Supplementary information, Figure S6A-S6D). PhyloP scores across 60 vertebrates49 generated by comparing the specific sites with random exonic regions of the same size indicated a significant conservation of the overlapping m6A and SRSF1- and 2-binding sites (P = 2.2e-16, Kolmogorov-Smirnov test; Supplementary information, Figure S7A). Furthermore, the potential SRSF1- or 2-recognized ESEs identified from m6A-modified exons by ESEfinder50,51 significantly overlapped with m6A sites. These predicted ESEs were also enriched with the RRACH motif found in m6A sites (Figure 4B). In details, 60%-80% of the predicted ESEs overlapped with m6A sites (P = 2.2e-16, Fisher test) and 20%-30% overlapped with the RRACH motif (P = 2.2e-16, Fisher test). Collectively, these results suggest that m6A might specifically influence the binding of SRSF1 and 2 to ESEs.


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)

m6A modification influences RNA binding ability of SRSF2. (A) Density plots showing the relative distance (X-axis), calculated by BEDTools' closestBed, between m6A sites and SRSF1-4-binding sites. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (B) The distance between the top-ranking SRSF1 and 2 ESEs predicted by ESEfinder and the nearest RRACH in identified m6A peak (red color) were calculated by BEDTools' closestBed (X axis). The distances of m6A-modified RRACH sites to the ESEs predicted by ESEfinder were calculated and their relative density distributions were shown as density plot. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (C) Analysis of the distribution of SRSF1-4-binding clusters along 5′ exon-intron and 3′ intron-exon boundaries (100 nt upstream and 300 nt downstream of 5′SS; 300 nt upstream and 100 nt downstream of 3′SS). (D) PAR-CLIP of SRSF2 in control and FTO-depleted 3T3-L1 cells transfected with the pCS2-Flag-SRSF2 plasmid. SRSF2 protein-RNA complex was pulled down with Flag M2 Affinity Gel (mouse), and then RNA was labeled and detected following instruction of biotin labeling kit. The expression levels of SRSF2 protein in whole cell lysate (WCE) was detected by western blotting with Flag antibody (rabbit). Knockdown efficiency of FTO was detected with its specific antibody. β-tubulin was used as loading control. (E) RNA binding ability of SRSF2 was calculated by normalizing binding RNAs to the corresponding pull-downed proteins. Statistical analysis shows the relative binding ability of SRSF2 in FTO-depleted cells (P < 0.0001) to that in control cells (siCTRL). See also Supplementary information, Figures S6-S7.
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fig4: m6A modification influences RNA binding ability of SRSF2. (A) Density plots showing the relative distance (X-axis), calculated by BEDTools' closestBed, between m6A sites and SRSF1-4-binding sites. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (B) The distance between the top-ranking SRSF1 and 2 ESEs predicted by ESEfinder and the nearest RRACH in identified m6A peak (red color) were calculated by BEDTools' closestBed (X axis). The distances of m6A-modified RRACH sites to the ESEs predicted by ESEfinder were calculated and their relative density distributions were shown as density plot. Randomly selected sites (blue color) within the same co-regulated genes were used as control. (C) Analysis of the distribution of SRSF1-4-binding clusters along 5′ exon-intron and 3′ intron-exon boundaries (100 nt upstream and 300 nt downstream of 5′SS; 300 nt upstream and 100 nt downstream of 3′SS). (D) PAR-CLIP of SRSF2 in control and FTO-depleted 3T3-L1 cells transfected with the pCS2-Flag-SRSF2 plasmid. SRSF2 protein-RNA complex was pulled down with Flag M2 Affinity Gel (mouse), and then RNA was labeled and detected following instruction of biotin labeling kit. The expression levels of SRSF2 protein in whole cell lysate (WCE) was detected by western blotting with Flag antibody (rabbit). Knockdown efficiency of FTO was detected with its specific antibody. β-tubulin was used as loading control. (E) RNA binding ability of SRSF2 was calculated by normalizing binding RNAs to the corresponding pull-downed proteins. Statistical analysis shows the relative binding ability of SRSF2 in FTO-depleted cells (P < 0.0001) to that in control cells (siCTRL). See also Supplementary information, Figures S6-S7.
Mentions: SR proteins are important for splice site recognition and intron processing. They recognize the pre-mRNA cis-acting elements, ESEs, and thereby participate in the selection of splice sites. ESEs recognized by SRSF1 and 2, like m6A, are highly enriched at the edges of exons44, suggesting a potential cooperative role between SRSF1/2 and m6A in RNA splicing. To test this, we calculated the distances between SRSF1-4-binding clusters based on the previously published CLIP-seq datasets45,46 and the nearest m6A sites. As a control, randomly selected sites with the same length as the m6A peak within the same exons were generated by BEDTools' shuffleBed. There was a significant spatial overlap between SRSF1- and 2-binding clusters and m6A sites (Figure 4A). In contrast, no overlap between SRSF3, 4 or ribonucleoproteins hnRNPC/hnRNRH47,48 and m6A sites was detected (Figure 4A and Supplementary information, Figure S6A-S6D). PhyloP scores across 60 vertebrates49 generated by comparing the specific sites with random exonic regions of the same size indicated a significant conservation of the overlapping m6A and SRSF1- and 2-binding sites (P = 2.2e-16, Kolmogorov-Smirnov test; Supplementary information, Figure S7A). Furthermore, the potential SRSF1- or 2-recognized ESEs identified from m6A-modified exons by ESEfinder50,51 significantly overlapped with m6A sites. These predicted ESEs were also enriched with the RRACH motif found in m6A sites (Figure 4B). In details, 60%-80% of the predicted ESEs overlapped with m6A sites (P = 2.2e-16, Fisher test) and 20%-30% overlapped with the RRACH motif (P = 2.2e-16, Fisher test). Collectively, these results suggest that m6A might specifically influence the binding of SRSF1 and 2 to ESEs.

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