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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 affects RNA splicing via modulating m6A. (A) The percentage of m6A-mRNAs in control (6 011 multi-isoform/2 699 single-isoform, P = 2.26e-5, Fisher test) and FTO-knockdown cells (6 916 multi-isoform/3 221 single-isoform, P = 8.46e-11, Fisher test) derived from single-isoform or multi-isoform genes compared to the distribution predicted by the Ensembl-annotation reference (background). (B) The distribution of m6A-containing exons across different categories of splicing events in both control (orange) and FTO-depleted samples (red) compared to that Ensembl-annotation reference (Blue). P value for each category was calculated between siCTRL and siFTO samples. **P < 0.01 (Student's t-test) is considered significant (n = 2). Results are shown as mean ± SD. CNE, constitutive exon; CE, cassette exon; A5SS, alternative 5′ splice site; ALE, alternative last exon; II, intron isoform; A3SS, alternative 3′ splice site; IR, intron retention; MXE, mutually exclusive exons; AFE, alternative first exon; EI, exon isoform. (C) 1 491 isoforms (1 335 genes) are co-regulated by FTO and METTL3. Arrows pointing up/down indicates up/down-regulation. (D) FTO depletion resulted in increased m6A levels in 522 isoforms (452 genes) of the 1 491 isoforms (1 335 genes) co-regulated by FTO and METTL3 (shown in C). (E) The heatmap shows the expression levels of 522 reverse-regulated isoforms by FTO and METTL3, as well as the m6A modification in FTO-depleted cells. (F) The heatmap shows m6A peaks in 5′-UTR, CDS, and 3′-UTR (522 isoforms co-regulated by FTO and METTL3) in control and FTO-deficient cells. Blue lines represent m6A peaks. Each horizontal line represents one gene. The number of new m6A peaks upon FTO depletion and new m6A peaks within a RRACH motif are shown below the heatmap. (G) Function enrichment analysis of 522 isoforms (452 genes) based on the DAVID GO analysis result. The cutoff parameters for enrichment analysis with Cytoscape software are: P < 0.005, FDR q < 0.1, overlap cutoff > 0.5. See also Supplementary information, Figures S2-S4.
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fig2: FTO affects RNA splicing via modulating m6A. (A) The percentage of m6A-mRNAs in control (6 011 multi-isoform/2 699 single-isoform, P = 2.26e-5, Fisher test) and FTO-knockdown cells (6 916 multi-isoform/3 221 single-isoform, P = 8.46e-11, Fisher test) derived from single-isoform or multi-isoform genes compared to the distribution predicted by the Ensembl-annotation reference (background). (B) The distribution of m6A-containing exons across different categories of splicing events in both control (orange) and FTO-depleted samples (red) compared to that Ensembl-annotation reference (Blue). P value for each category was calculated between siCTRL and siFTO samples. **P < 0.01 (Student's t-test) is considered significant (n = 2). Results are shown as mean ± SD. CNE, constitutive exon; CE, cassette exon; A5SS, alternative 5′ splice site; ALE, alternative last exon; II, intron isoform; A3SS, alternative 3′ splice site; IR, intron retention; MXE, mutually exclusive exons; AFE, alternative first exon; EI, exon isoform. (C) 1 491 isoforms (1 335 genes) are co-regulated by FTO and METTL3. Arrows pointing up/down indicates up/down-regulation. (D) FTO depletion resulted in increased m6A levels in 522 isoforms (452 genes) of the 1 491 isoforms (1 335 genes) co-regulated by FTO and METTL3 (shown in C). (E) The heatmap shows the expression levels of 522 reverse-regulated isoforms by FTO and METTL3, as well as the m6A modification in FTO-depleted cells. (F) The heatmap shows m6A peaks in 5′-UTR, CDS, and 3′-UTR (522 isoforms co-regulated by FTO and METTL3) in control and FTO-deficient cells. Blue lines represent m6A peaks. Each horizontal line represents one gene. The number of new m6A peaks upon FTO depletion and new m6A peaks within a RRACH motif are shown below the heatmap. (G) Function enrichment analysis of 522 isoforms (452 genes) based on the DAVID GO analysis result. The cutoff parameters for enrichment analysis with Cytoscape software are: P < 0.005, FDR q < 0.1, overlap cutoff > 0.5. See also Supplementary information, Figures S2-S4.

Mentions: Previous findings have shown that m6A demethylases and members of the m6A methyltransferase complex are localized in nuclear speckles, suggesting a potential role of m6A in RNA splicing9,27,34,39. Analyses by Cufflink showed that 279 out of 694 genes without obvious changes in gene expression levels (fold change < 2) but with significant changes in the main isoforms (fold change > 2), were m6A-modified (P = 2.2e-10, hypergeometric test). Scripture analysis showed that 6 329 of 7 374 genes with differently spliced isoforms were m6A-modified (P < 1e-305, hypergeometric test). When comparing single- and multi-isoform genes with the Ensembl-annotated reference (background), it was evident that m6A is more common in multi-isoform genes compared to single-isoform genes (Figure 2A). In addition, the average number of m6A peaks per gene was also higher in multi-isoform genes (4.52 peaks per gene) compared to single-isoform genes (3.65 peaks per gene). At the exon/intron level, 376 of 798 differentially expressed exons (P = 3.9e-27, hypergeometric test) and 1 613 of 3 521 differentially expressed introns (P = 2.6e-105, hypergeometric test) bare m6A. Comparing the m6A-modified exons with Ensembl-annotated genomic exons, we found that 51% of the constitutive exons (CNE) were m6A-modified, which is 14% less than the 65% predicted by the Ensembl-annotation (P < 2.2e-16, Fisher's exact test). In contrast, m6A was overrepresented in both alternative cassette (CE) exons and intron retention (IR) splicing events, and m6A peaks within CE exons increased upon FTO depletion (P < 0.01, t-test) (Figure 2B), suggesting that FTO directly regulates m6A levels during this type of splicing.


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 affects RNA splicing via modulating m6A. (A) The percentage of m6A-mRNAs in control (6 011 multi-isoform/2 699 single-isoform, P = 2.26e-5, Fisher test) and FTO-knockdown cells (6 916 multi-isoform/3 221 single-isoform, P = 8.46e-11, Fisher test) derived from single-isoform or multi-isoform genes compared to the distribution predicted by the Ensembl-annotation reference (background). (B) The distribution of m6A-containing exons across different categories of splicing events in both control (orange) and FTO-depleted samples (red) compared to that Ensembl-annotation reference (Blue). P value for each category was calculated between siCTRL and siFTO samples. **P < 0.01 (Student's t-test) is considered significant (n = 2). Results are shown as mean ± SD. CNE, constitutive exon; CE, cassette exon; A5SS, alternative 5′ splice site; ALE, alternative last exon; II, intron isoform; A3SS, alternative 3′ splice site; IR, intron retention; MXE, mutually exclusive exons; AFE, alternative first exon; EI, exon isoform. (C) 1 491 isoforms (1 335 genes) are co-regulated by FTO and METTL3. Arrows pointing up/down indicates up/down-regulation. (D) FTO depletion resulted in increased m6A levels in 522 isoforms (452 genes) of the 1 491 isoforms (1 335 genes) co-regulated by FTO and METTL3 (shown in C). (E) The heatmap shows the expression levels of 522 reverse-regulated isoforms by FTO and METTL3, as well as the m6A modification in FTO-depleted cells. (F) The heatmap shows m6A peaks in 5′-UTR, CDS, and 3′-UTR (522 isoforms co-regulated by FTO and METTL3) in control and FTO-deficient cells. Blue lines represent m6A peaks. Each horizontal line represents one gene. The number of new m6A peaks upon FTO depletion and new m6A peaks within a RRACH motif are shown below the heatmap. (G) Function enrichment analysis of 522 isoforms (452 genes) based on the DAVID GO analysis result. The cutoff parameters for enrichment analysis with Cytoscape software are: P < 0.005, FDR q < 0.1, overlap cutoff > 0.5. See also Supplementary information, Figures S2-S4.
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fig2: FTO affects RNA splicing via modulating m6A. (A) The percentage of m6A-mRNAs in control (6 011 multi-isoform/2 699 single-isoform, P = 2.26e-5, Fisher test) and FTO-knockdown cells (6 916 multi-isoform/3 221 single-isoform, P = 8.46e-11, Fisher test) derived from single-isoform or multi-isoform genes compared to the distribution predicted by the Ensembl-annotation reference (background). (B) The distribution of m6A-containing exons across different categories of splicing events in both control (orange) and FTO-depleted samples (red) compared to that Ensembl-annotation reference (Blue). P value for each category was calculated between siCTRL and siFTO samples. **P < 0.01 (Student's t-test) is considered significant (n = 2). Results are shown as mean ± SD. CNE, constitutive exon; CE, cassette exon; A5SS, alternative 5′ splice site; ALE, alternative last exon; II, intron isoform; A3SS, alternative 3′ splice site; IR, intron retention; MXE, mutually exclusive exons; AFE, alternative first exon; EI, exon isoform. (C) 1 491 isoforms (1 335 genes) are co-regulated by FTO and METTL3. Arrows pointing up/down indicates up/down-regulation. (D) FTO depletion resulted in increased m6A levels in 522 isoforms (452 genes) of the 1 491 isoforms (1 335 genes) co-regulated by FTO and METTL3 (shown in C). (E) The heatmap shows the expression levels of 522 reverse-regulated isoforms by FTO and METTL3, as well as the m6A modification in FTO-depleted cells. (F) The heatmap shows m6A peaks in 5′-UTR, CDS, and 3′-UTR (522 isoforms co-regulated by FTO and METTL3) in control and FTO-deficient cells. Blue lines represent m6A peaks. Each horizontal line represents one gene. The number of new m6A peaks upon FTO depletion and new m6A peaks within a RRACH motif are shown below the heatmap. (G) Function enrichment analysis of 522 isoforms (452 genes) based on the DAVID GO analysis result. The cutoff parameters for enrichment analysis with Cytoscape software are: P < 0.005, FDR q < 0.1, overlap cutoff > 0.5. See also Supplementary information, Figures S2-S4.
Mentions: Previous findings have shown that m6A demethylases and members of the m6A methyltransferase complex are localized in nuclear speckles, suggesting a potential role of m6A in RNA splicing9,27,34,39. Analyses by Cufflink showed that 279 out of 694 genes without obvious changes in gene expression levels (fold change < 2) but with significant changes in the main isoforms (fold change > 2), were m6A-modified (P = 2.2e-10, hypergeometric test). Scripture analysis showed that 6 329 of 7 374 genes with differently spliced isoforms were m6A-modified (P < 1e-305, hypergeometric test). When comparing single- and multi-isoform genes with the Ensembl-annotated reference (background), it was evident that m6A is more common in multi-isoform genes compared to single-isoform genes (Figure 2A). In addition, the average number of m6A peaks per gene was also higher in multi-isoform genes (4.52 peaks per gene) compared to single-isoform genes (3.65 peaks per gene). At the exon/intron level, 376 of 798 differentially expressed exons (P = 3.9e-27, hypergeometric test) and 1 613 of 3 521 differentially expressed introns (P = 2.6e-105, hypergeometric test) bare m6A. Comparing the m6A-modified exons with Ensembl-annotated genomic exons, we found that 51% of the constitutive exons (CNE) were m6A-modified, which is 14% less than the 65% predicted by the Ensembl-annotation (P < 2.2e-16, Fisher's exact test). In contrast, m6A was overrepresented in both alternative cassette (CE) exons and intron retention (IR) splicing events, and m6A peaks within CE exons increased upon FTO depletion (P < 0.01, t-test) (Figure 2B), suggesting that FTO directly regulates m6A levels during this type of splicing.

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