TTP protects against acute liver failure by regulating CCL2 and CCL5 through m6A RNA methylation
Pingping Xiao, Mingxuan Li, Mengsi Zhou, Xuejun Zhao, Cheng Wang, Jinglin Qiu, Qian Fang, Hong Jiang, Huifen Dong, Rui Zhou
Pingping Xiao, Mingxuan Li, Mengsi Zhou, Xuejun Zhao, Cheng Wang, Jinglin Qiu, Qian Fang, Hong Jiang, Huifen Dong, Rui Zhou
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Research Article Hepatology Inflammation

TTP protects against acute liver failure by regulating CCL2 and CCL5 through m6A RNA methylation

Abstract

Tristetraprolin (TTP), an important immunosuppressive protein regulating mRNA decay through recognition of the AU-rich elements (AREs) within the 3′-UTRs of mRNAs, participates in the pathogenesis of liver diseases. However, whether TTP regulates mRNA stability through other mechanisms remains poorly understood. Here, we report that TTP was upregulated in acute liver failure (ALF), resulting in decreased mRNA stabilities of CCL2 and CCL5 through promotion of N6-methyladenosine (m6A) mRNA methylation. Overexpression of TTP could markedly ameliorate hepatic injury in vivo. TTP regulated the mRNA stabilization of CCL2 and CCL5. Interestingly, increased m6A methylation in CCL2 and CCL5 mRNAs promoted TTP-mediated RNA destabilization. Moreover, induction of TTP upregulated expression levels of WT1 associated protein, methyltransferase like 14, and YT521-B homology N6-methyladenosine RNA binding protein 2, which encode enzymes regulating m6A methylation, resulting in a global increase of m6A methylation and amelioration of liver injury due to enhanced degradation of CCL2 and CCL5. These findings suggest a potentially novel mechanism by which TTP modulates mRNA stabilities of CCL2 and CCL5 through m6A RNA methylation, which is involved in the pathogenesis of ALF.

Authors

Pingping Xiao, Mingxuan Li, Mengsi Zhou, Xuejun Zhao, Cheng Wang, Jinglin Qiu, Qian Fang, Hong Jiang, Huifen Dong, Rui Zhou

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Figure 6

TTP affects m6A-mediated posttranscription of CCL2 and CCL5 through regulation of WTAP, METTL14, and YTHDF2.

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TTP affects m6A-mediated posttranscription of CCL2 and CCL5 through regu...
(A) The level of CCL2 was analyzed by real-time PCR in WTAP, METTL14, or YTHDF2 knockdown cells transfected with TTP-overexpression vector. (B) TTP-overexpression cells were cotransfected with the luciferase construct containing the CDS of CCL2 (pMIR-GLO-CCL2-CDS) and WTAP, METTL14, or YTHDF2 shRNA, followed by luciferase analysis. (C) The interactions between TTP and YTHDF2 were analyzed by Co-IP in HL7702 cells. (D) RIP was used to detect the physical association between TTP and CCL2 or CCL5 mRNAs in HL7702 cells. (E) RIP was used to detect the physical association between YTHDF2 and CCL2 or CCL5 mRNAs in YTHDF2-knockdown cells. (F) The stabilities of CCL2 and CCL5 mRNA were calculated in TTP-knockdown cells transfected with YTHDF2-overexpression plasmid. The pxj (empty vector) acted as a control for pxj YTHDF2. (G) iCLIP-PCR was conducted to detect TTP binding positions in m6A sites of CCL2 and CCL5 mRNAs in TTP-knockdown cells. Data represent mean ± SEM from 3 independent experiments. Statistics by 2-way ANOVA with Tukey’s multiple comparisons test (A and B), 2-tailed Student’s t test (D), 1-way ANOVA with Tukey’s multiple comparisons test (E and G), and 2-way repeated measures ANOVA with Tukey’s multiple comparisons test (F). *P < 0.05 versus Ctrl.