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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 is enriched at exonic splice sites. (A-D) Schematic analysis of the distribution of m6A peaks (upper panel in each figure; 100 nt upstream and 300 nt downstream from 5′SS; 300 nt upstream and 100 nt downstream from 3′SS) and m6A enrichment (lower panel in each figure; 100 nt up/downstream of the 5′ SS or 3′ SS) along 5′ exon-intron and 3′ intron-exon boundaries in control (blue) and FTO-deficient (red) cells. (A, C) m6A density in the vicinity of constitutive SS (A) or alternative SS (C) in the 5 326 genes showing changes in isoform expression upon FTO depletion. (B, D) Similar to A and C but here the m6A density was determined in the 452 FTO target genes showing increased m6A levels after FTO knockdown. The m6A levels in each region between control and FTO depletion are shown as mean ± SD and statistical analysis on their difference were performed by Student's t-test. **P < 0.01. See also Supplementary information, Figures S4 and S5.
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fig3: m6A is enriched at exonic splice sites. (A-D) Schematic analysis of the distribution of m6A peaks (upper panel in each figure; 100 nt upstream and 300 nt downstream from 5′SS; 300 nt upstream and 100 nt downstream from 3′SS) and m6A enrichment (lower panel in each figure; 100 nt up/downstream of the 5′ SS or 3′ SS) along 5′ exon-intron and 3′ intron-exon boundaries in control (blue) and FTO-deficient (red) cells. (A, C) m6A density in the vicinity of constitutive SS (A) or alternative SS (C) in the 5 326 genes showing changes in isoform expression upon FTO depletion. (B, D) Similar to A and C but here the m6A density was determined in the 452 FTO target genes showing increased m6A levels after FTO knockdown. The m6A levels in each region between control and FTO depletion are shown as mean ± SD and statistical analysis on their difference were performed by Student's t-test. **P < 0.01. See also Supplementary information, Figures S4 and S5.

Mentions: Cis-elements found within exons, such as exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs) in the vicinity of splice sites (both 5′ and 3′ splice sites), play important roles in regulating inclusion or skipping of exons40. In addition, in human and mouse the conserved intronic sequences flanking both sides of an alternatively spliced exon have also been proposed to be involved in the regulation of alternative splicing41,42. The findings presented so far in combination with previous reports suggest the possible involvement of m6A in the regulation of RNA splicing18,27. To further test this, we examined the distribution of m6A sites in the vicinity of splice sites. Using procedures reported previously43, we analyzed m6A peak distributions in mRNAs within 100-300 nt upstream or downstream of the constitutive splice sites. Meanwhile, to analyze the effect of FTO depletion on m6A peak number and enrichment, total m6A enrichments for m6A peaks located within 100 nt up- and downstream of 5′ and 3′ splice sites were calculated, respectively. For 5 326 genes showing changes in isoform expression upon FTO depletion, a significant overrepresentation of m6A sites in 5′ and 3′ exonic sequences flanking the constitutive splice site was observed when compared to intronic sequences adjacent to the splice site (Figure 3A and Supplementary information, Table S3). FTO deficiency further increased the m6A enrichment in these regions (Figure 3A, lower panel and Supplementary information, Table S3). For the 522 isoforms (452 genes) that were inversely affected in expression levels by FTO and METTL3 depletion and showed increased m6A levels upon FTO depletion, the effect of FTO depletion on m6A levels in regions surrounding constitutive splice sites was even more pronounced (5′ exon, 2.50-fold; 3′ exon, 2.68-fold; Figure 3B, lower panel). Similar results regarding m6A distribution pattern and enrichment were obtained around alternative splice sites (Figure 3C and 3D). On the whole, although FTO depletion barely affected the overall distribution pattern of m6A peaks, it increased the m6A levels at individual m6A peaks (Figure 3A-3D). We next analyzed previously published m6A-Seq/MeRIP-Seq datasets17,18. Consistently, in these datasets the majority of m6A peaks in exons were also mainly located in regions adjacent to constitutive and alternative splice sites (Supplementary information, Figure S5A).


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 is enriched at exonic splice sites. (A-D) Schematic analysis of the distribution of m6A peaks (upper panel in each figure; 100 nt upstream and 300 nt downstream from 5′SS; 300 nt upstream and 100 nt downstream from 3′SS) and m6A enrichment (lower panel in each figure; 100 nt up/downstream of the 5′ SS or 3′ SS) along 5′ exon-intron and 3′ intron-exon boundaries in control (blue) and FTO-deficient (red) cells. (A, C) m6A density in the vicinity of constitutive SS (A) or alternative SS (C) in the 5 326 genes showing changes in isoform expression upon FTO depletion. (B, D) Similar to A and C but here the m6A density was determined in the 452 FTO target genes showing increased m6A levels after FTO knockdown. The m6A levels in each region between control and FTO depletion are shown as mean ± SD and statistical analysis on their difference were performed by Student's t-test. **P < 0.01. See also Supplementary information, Figures S4 and S5.
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Related In: Results  -  Collection

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fig3: m6A is enriched at exonic splice sites. (A-D) Schematic analysis of the distribution of m6A peaks (upper panel in each figure; 100 nt upstream and 300 nt downstream from 5′SS; 300 nt upstream and 100 nt downstream from 3′SS) and m6A enrichment (lower panel in each figure; 100 nt up/downstream of the 5′ SS or 3′ SS) along 5′ exon-intron and 3′ intron-exon boundaries in control (blue) and FTO-deficient (red) cells. (A, C) m6A density in the vicinity of constitutive SS (A) or alternative SS (C) in the 5 326 genes showing changes in isoform expression upon FTO depletion. (B, D) Similar to A and C but here the m6A density was determined in the 452 FTO target genes showing increased m6A levels after FTO knockdown. The m6A levels in each region between control and FTO depletion are shown as mean ± SD and statistical analysis on their difference were performed by Student's t-test. **P < 0.01. See also Supplementary information, Figures S4 and S5.
Mentions: Cis-elements found within exons, such as exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs) in the vicinity of splice sites (both 5′ and 3′ splice sites), play important roles in regulating inclusion or skipping of exons40. In addition, in human and mouse the conserved intronic sequences flanking both sides of an alternatively spliced exon have also been proposed to be involved in the regulation of alternative splicing41,42. The findings presented so far in combination with previous reports suggest the possible involvement of m6A in the regulation of RNA splicing18,27. To further test this, we examined the distribution of m6A sites in the vicinity of splice sites. Using procedures reported previously43, we analyzed m6A peak distributions in mRNAs within 100-300 nt upstream or downstream of the constitutive splice sites. Meanwhile, to analyze the effect of FTO depletion on m6A peak number and enrichment, total m6A enrichments for m6A peaks located within 100 nt up- and downstream of 5′ and 3′ splice sites were calculated, respectively. For 5 326 genes showing changes in isoform expression upon FTO depletion, a significant overrepresentation of m6A sites in 5′ and 3′ exonic sequences flanking the constitutive splice site was observed when compared to intronic sequences adjacent to the splice site (Figure 3A and Supplementary information, Table S3). FTO deficiency further increased the m6A enrichment in these regions (Figure 3A, lower panel and Supplementary information, Table S3). For the 522 isoforms (452 genes) that were inversely affected in expression levels by FTO and METTL3 depletion and showed increased m6A levels upon FTO depletion, the effect of FTO depletion on m6A levels in regions surrounding constitutive splice sites was even more pronounced (5′ exon, 2.50-fold; 3′ exon, 2.68-fold; Figure 3B, lower panel). Similar results regarding m6A distribution pattern and enrichment were obtained around alternative splice sites (Figure 3C and 3D). On the whole, although FTO depletion barely affected the overall distribution pattern of m6A peaks, it increased the m6A levels at individual m6A peaks (Figure 3A-3D). We next analyzed previously published m6A-Seq/MeRIP-Seq datasets17,18. Consistently, in these datasets the majority of m6A peaks in exons were also mainly located in regions adjacent to constitutive and alternative splice sites (Supplementary information, Figure S5A).

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