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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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Cooperative role of m6A in regulating SRSF2 function at splice sites. The splicing factor SRSF2 can recognize exon splicing enhancer (ESE), inducing exon inclusion. METTL3-mediated m6A modification enhances the recruitment of SRSF2 to its target ESE, promoting exon inclusion. On the other hand demethylation of m6A-RRACHs near ESEs by FTO prevents recognition of the ESEs by SRSF2, thereby inhibiting inclusion and instead promoting exon skipping of the specific exon. M6A co-regulated by methyltransferases and demthylases serves as a new RNA splicing exonic cis-regulatory element that in combination with ESEs regulates SRSF2 protein recruitment.
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fig7: Cooperative role of m6A in regulating SRSF2 function at splice sites. The splicing factor SRSF2 can recognize exon splicing enhancer (ESE), inducing exon inclusion. METTL3-mediated m6A modification enhances the recruitment of SRSF2 to its target ESE, promoting exon inclusion. On the other hand demethylation of m6A-RRACHs near ESEs by FTO prevents recognition of the ESEs by SRSF2, thereby inhibiting inclusion and instead promoting exon skipping of the specific exon. M6A co-regulated by methyltransferases and demthylases serves as a new RNA splicing exonic cis-regulatory element that in combination with ESEs regulates SRSF2 protein recruitment.

Mentions: In this study we demonstrate that FTO contributes to the regulation of mRNA alternative splicing by modulating m6A levels and hence SRSF2 binding at splice sites. We show a novel FTO-dependent function of m6A in the regulation of mRNA splicing in adipocytes and in the process of adipogenesis. There are several pieces of evidence suggesting a role of m6A in RNA splicing: m6A are preferentially found in exons and UTR regions in multi-isoform genes and alternatively spliced exons18, binding of the m6A methyltransferase complex is especially enriched in genes with multiple isoforms and alternatively spliced exon junctions27, and finally, depletion of either METTL3 or ALKBH5 causes mRNA splicing alterations18,34. However, it has remained unclear how m6A may contribute to the regulation of mRNA alternative splicing. The pronounced enrichment of m6A in exonic sequences spatially overlapping with ESEs and binding clusters of specific SR proteins, together with the finding that m6A regulates RNA binding of SRSF2, suggests that m6A might influence the choice of exon inclusion/exclusion by regulating SRSF2 access to its targets. The accurate recognition of exon/intron boundaries is essential for correct splicing and generation of mature mRNA. In higher eukaryotes, exon-intron junctions are defined by conserved intronic cis-elements, including the 5′ splice site and 3′ splice site. Exonic cis-elements such as exonic splicing enhancers (ESEs) or silencers (ESSs) are important for correct splice-site identification and prevalent in most exons, including constitutive ones. ESEs serve as binding sites for specific serine/arginine-rich (SR) proteins. ESEs and their binding SR proteins frequently exist in protein-coding exons, compared with flanking intronic regions53. Similarly, in CDS regions, m6A is highly enriched at the edges of exons adjacent to splice sites relative to intron regions. Knockdown of FTO specifically affects the RNA binding ability of SRSF2, but not that of SRSF4. This might not be surprising since SRSF2 readily binds to exons in contrast to other SR proteins such as SRSF3 and SRSF4 which have been reported to preferentially bind to intronless genes, ncRNAs and introns or 3′-UTRs of intron-containing genes45,46,54. Hence m6A dynamics appears to specifically affect the RNA binding ability of SRSF2, thus specifically influencing the splicing outcome of genes under the regulation of SRSF2 (Figures 5 and 6), which can promote both inclusion and skipping of exons45. However, in the case of m6A-dependent SRSF2 regulation of mRNA splicing, SRSF2 appears to exclusively promote constitutive splicing (inclusion of exons) while inhibiting alternative splicing (exclusion of exons). Thus m6A seems to bring another layer of regulation to the mRNA splicing process (Figure 7). A thorough mechanistic understanding of the interplay between m6A, ESEs and SR proteins will be a challenge for future studies.


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)

Cooperative role of m6A in regulating SRSF2 function at splice sites. The splicing factor SRSF2 can recognize exon splicing enhancer (ESE), inducing exon inclusion. METTL3-mediated m6A modification enhances the recruitment of SRSF2 to its target ESE, promoting exon inclusion. On the other hand demethylation of m6A-RRACHs near ESEs by FTO prevents recognition of the ESEs by SRSF2, thereby inhibiting inclusion and instead promoting exon skipping of the specific exon. M6A co-regulated by methyltransferases and demthylases serves as a new RNA splicing exonic cis-regulatory element that in combination with ESEs regulates SRSF2 protein recruitment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Cooperative role of m6A in regulating SRSF2 function at splice sites. The splicing factor SRSF2 can recognize exon splicing enhancer (ESE), inducing exon inclusion. METTL3-mediated m6A modification enhances the recruitment of SRSF2 to its target ESE, promoting exon inclusion. On the other hand demethylation of m6A-RRACHs near ESEs by FTO prevents recognition of the ESEs by SRSF2, thereby inhibiting inclusion and instead promoting exon skipping of the specific exon. M6A co-regulated by methyltransferases and demthylases serves as a new RNA splicing exonic cis-regulatory element that in combination with ESEs regulates SRSF2 protein recruitment.
Mentions: In this study we demonstrate that FTO contributes to the regulation of mRNA alternative splicing by modulating m6A levels and hence SRSF2 binding at splice sites. We show a novel FTO-dependent function of m6A in the regulation of mRNA splicing in adipocytes and in the process of adipogenesis. There are several pieces of evidence suggesting a role of m6A in RNA splicing: m6A are preferentially found in exons and UTR regions in multi-isoform genes and alternatively spliced exons18, binding of the m6A methyltransferase complex is especially enriched in genes with multiple isoforms and alternatively spliced exon junctions27, and finally, depletion of either METTL3 or ALKBH5 causes mRNA splicing alterations18,34. However, it has remained unclear how m6A may contribute to the regulation of mRNA alternative splicing. The pronounced enrichment of m6A in exonic sequences spatially overlapping with ESEs and binding clusters of specific SR proteins, together with the finding that m6A regulates RNA binding of SRSF2, suggests that m6A might influence the choice of exon inclusion/exclusion by regulating SRSF2 access to its targets. The accurate recognition of exon/intron boundaries is essential for correct splicing and generation of mature mRNA. In higher eukaryotes, exon-intron junctions are defined by conserved intronic cis-elements, including the 5′ splice site and 3′ splice site. Exonic cis-elements such as exonic splicing enhancers (ESEs) or silencers (ESSs) are important for correct splice-site identification and prevalent in most exons, including constitutive ones. ESEs serve as binding sites for specific serine/arginine-rich (SR) proteins. ESEs and their binding SR proteins frequently exist in protein-coding exons, compared with flanking intronic regions53. Similarly, in CDS regions, m6A is highly enriched at the edges of exons adjacent to splice sites relative to intron regions. Knockdown of FTO specifically affects the RNA binding ability of SRSF2, but not that of SRSF4. This might not be surprising since SRSF2 readily binds to exons in contrast to other SR proteins such as SRSF3 and SRSF4 which have been reported to preferentially bind to intronless genes, ncRNAs and introns or 3′-UTRs of intron-containing genes45,46,54. Hence m6A dynamics appears to specifically affect the RNA binding ability of SRSF2, thus specifically influencing the splicing outcome of genes under the regulation of SRSF2 (Figures 5 and 6), which can promote both inclusion and skipping of exons45. However, in the case of m6A-dependent SRSF2 regulation of mRNA splicing, SRSF2 appears to exclusively promote constitutive splicing (inclusion of exons) while inhibiting alternative splicing (exclusion of exons). Thus m6A seems to bring another layer of regulation to the mRNA splicing process (Figure 7). A thorough mechanistic understanding of the interplay between m6A, ESEs and SR proteins will be a challenge for future studies.

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