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Background <p>N6-methyladenosine (m6A) modification of mRNAs through METTL3 maintains homeostasis in many cell types. The mechanism by which METTL3 complexes together with reader proteins, especially the YTH domain-containing protein, governs endothelial homeostasis is not yet fully understood.</p> Methods and results <p>Primary endothelial cells (HUVECs), EA.hy926 cells, and rat aortas were used to modulate METTL3 activity and assess gene expression changes by molecular and biochemical assays. m6A RNA immunoprecipitation and RNA–protein interaction analyses were performed to determine transcript-specific m6A modification and YTHDF-mediated regulation. Interplaying with METTL3 caused abrupt yet selective loss of eNOS gene expression while increasing inflammatory adhesion molecule gene expression in cultured endothelial cells (EC) and rat aorta. Interestingly, many other genes associated with endothelial function/inflammation/senescence including CD31, CD144, KLF2, p65, and p53 remained unaltered upon METTL3 inhbition. MeRIP analysis revealed significant m6A modifications of several gene transcript including eNOS, and inflammatory adhesion molecules. m6A modification of gene transcripts selectively stabilized eNOS while causing degradation of inflammatory adhesion molecules without affecting the stability of other genes such as p65. Interestingly, RIP analysis showed that YTHDF1 was preferentially bound to m6A-modified eNOS while YTHDF2 was selectively associated with inflammatory adhesion molecules, causing differential regulation. Moreover, we failed to detect association of YTHDF1 or YTHDF2 to m6A modified p65 and KLF2 transcript.</p> Conclusion <p>Taken together, the current study describes the essential role of the METTL3 complex in maintaining endothelial homeostasis through differential association of m6A modified gene transcripts with the reader proteins.</p>

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YTHDF proteins differentially regulate a subset of m6A-modified transcripts to restrain endothelial inflammation during homeostasis

  • 期刊论文

  • 发表日期：

    2026年3月18日

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  • Ramakrishnan Shyam Kumar,
  • Srinjoy Chakraborty,
  • Hariharan Jayakumar,
  • Manasa Bharath,
  • Niyati Pandya Thakkar,
  • Syamantak Majumder

摘要

Background

N6-methyladenosine (m6A) modification of mRNAs through METTL3 maintains homeostasis in many cell types. The mechanism by which METTL3 complexes together with reader proteins, especially the YTH domain-containing protein, governs endothelial homeostasis is not yet fully understood.

Methods and results

Primary endothelial cells (HUVECs), EA.hy926 cells, and rat aortas were used to modulate METTL3 activity and assess gene expression changes by molecular and biochemical assays. m6A RNA immunoprecipitation and RNA–protein interaction analyses were performed to determine transcript-specific m6A modification and YTHDF-mediated regulation. Interplaying with METTL3 caused abrupt yet selective loss of eNOS gene expression while increasing inflammatory adhesion molecule gene expression in cultured endothelial cells (EC) and rat aorta. Interestingly, many other genes associated with endothelial function/inflammation/senescence including CD31, CD144, KLF2, p65, and p53 remained unaltered upon METTL3 inhbition. MeRIP analysis revealed significant m6A modifications of several gene transcript including eNOS, and inflammatory adhesion molecules. m6A modification of gene transcripts selectively stabilized eNOS while causing degradation of inflammatory adhesion molecules without affecting the stability of other genes such as p65. Interestingly, RIP analysis showed that YTHDF1 was preferentially bound to m6A-modified eNOS while YTHDF2 was selectively associated with inflammatory adhesion molecules, causing differential regulation. Moreover, we failed to detect association of YTHDF1 or YTHDF2 to m6A modified p65 and KLF2 transcript.

Conclusion

Taken together, the current study describes the essential role of the METTL3 complex in maintaining endothelial homeostasis through differential association of m6A modified gene transcripts with the reader proteins.