Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models
Yong Ge, … , Shaochun Yuan, Anlong Xu
Yong Ge, … , Shaochun Yuan, Anlong Xu
Published July 15, 2024
Citation Information: J Clin Invest. 2024;134(14):e177932. https://doi.org/10.1172/JCI177932.
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Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models

Abstract

Emerging evidence has linked the dysregulation of N6-methyladenosine (m6A) modification to inflammation and inflammatory diseases, but the underlying mechanism still needs investigation. Here, we found that high levels of m6A modification in a variety of hyperinflammatory states are p65-dependent because Wilms tumor 1–associated protein (WTAP), a key component of the “writer” complex, is transcriptionally regulated by p65, and its overexpression can lead to increased levels of m6A modification. Mechanistically, upregulated WTAP is more prone to phase separation to facilitate the aggregation of the writer complex to nuclear speckles and the deposition of m6A marks on transcriptionally active inflammatory transcripts, thereby accelerating the proinflammatory response. Further, a myeloid deficiency in WTAP attenuates the severity of LPS-induced sepsis and DSS-induced IBD. Thus, the proinflammatory effect of WTAP is a general risk-increasing mechanism, and interrupting the assembly of the m6A writer complex to reduce the global m6A levels by targeting the phase separation of WTAP may be a potential and promising therapeutic strategy for alleviating hyperinflammation.

Authors

Yong Ge, Rong Chen, Tao Ling, Biaodi Liu, Jingrong Huang, Youxiang Cheng, Yi Lin, Hongxuan Chen, Xiongmei Xie, Guomeng Xia, Guanzheng Luo, Shaochun Yuan, Anlong Xu

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Figure 6

Phase separation of WTAP leads to METTL3 recruitment to efficiently modify inflammatory transcripts.

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Phase separation of WTAP leads to METTL3 recruitment to efficiently modi...
(A and B) Prediction of the disordered regions and prion-like domains (PrD) of WTAP using the IUPred2A (A) or PLAAC (B) tool. (C) Images of GFP-WTAP droplet formation at room temperature with indicated GFP-WTAP concentrations (350 mM NaCl). Scale bars: 5 μm. (D) LSCM images of 293T cells expressing PEGFP-N1 or GFP-tagged WTAP constructs, and dotted box in left-most image is enlarged in the other images. Scale bars: 20 or 5 μm. (E) FRAP assays of the GFP-tagged WTAP droplets before and after photobleaching, and the dotted circle highlights the foci undergoing targeted bleaching. Scale bars: 10 μm. (F) FRAP quantification of GFP-WTAP droplets over a period of 65 seconds. (G) LSCM images of HeLa cells treated with TNF-α for 2 hours, and dotted box in left-most image is enlarged in the other images. Scale bars: 20 or 5 μm. (H) Statistics of the number of WTAP droplets in G. (I) Quantitative line profile of colocalization along a white arrow of the image of G. (J) Statistics of the number of SC35 droplets in G. (K) LSCM images of 293T cells expressing full-length or truncated WTAP labeled with GFP. Scale bars: 20 or 5 μm. (L) LSCM images of the mCherry-tagged METTL3 droplets (red) in 293T cells before and after cotransfection with GFP-tagged full-length or truncated WTAP. Scale bars: 10 or 5 μm. (M)FRAP assays of the mCherry-tagged METTL3 droplets before and after photobleaching from L. The dotted circle highlights the foci undergoing targeted bleaching. Scale bars: 10 μm. (N) FRAP quantification of mCherry-METTL3 droplets over a period of 100 seconds. (O) METTL3 was immunoprecipitated and RIP-qPCR was performed to assess the association of IL6ST transcripts with METTL3. (P) Working model for WTAP facilitating inflammatory responses through m6A modification and phase separation. Data are representative of 3 independent biological experiments in C–E, G, and K–M. Data are presented as the mean ± SD in H, J and O, with individual measurements overlaid as dots. Statistical analysis was performed using 2-tailed Student’s t test in H and J, or 1-way ANOVA multiple comparisons in O. Indicated scale bars are shown in C–E, G, and K–M.