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Acute depletion of METTL3 implicates N ⁶-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

ABSTRACT: RNA N ⁶-methyladenosine (m⁶A) modification plays important roles in multiple aspects of RNA regulation. m⁶A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m⁶A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m⁶A peaks are located in alternative introns/exons, often close to 5' splice sites. m⁶A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m⁶A peaks. For terminal or variable-length exons, m⁶A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m⁶A and upstream of a 5' splice site that is promoted in the presence of m⁶A. Genes with the most immediate effects on splicing include several components of the m⁶A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m⁶A machinery and the regulation of RNA splicing.

SUBMITTER: Wei G

PROVIDER: S-EPMC8327914 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundN6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known.MethodsTo determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function.ResultsWe measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis.ConclusionsOur study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

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Acute depletion of METTL3 implicates N ⁶-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Acute depletion of METTL3 implicates N 6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Acute depletion of METTL3 implicates N ⁶-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.