METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on ...METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell(mESC).While comparable global hypo-methylation in RNA m^(6)A was observed in Mettl3 or Mettl14 knockout mESCs,respectively.Mettl14 knockout led to a globally decreased nascent RNA synthesis,whereas Mettl3 depletion resulted in transcription upregulation,suggesting that METTL14 might possess an mA-independent role in gene regulation.We found that METTL14 colocalizes with the repressive H3K27me3 modification.Mechanistically,METTL14,but not METTL3,binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3.Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression.The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs.This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m^(6)A,and critically impacts transcriptional regulation,stemness maintenance,and differentiation ofmESCs.展开更多
目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6...目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6、iNO、Arg-1和CD206表达变化以及PI3K/AKT/GSK3β/β-catenin信号通路相关蛋白表达情况。随后加入PI3K/AKT/GSK3β/β-catenin信号通路激动剂和抑制剂,检测过表达或抑制METTL14后,巨噬细胞IL-6、iNO、Arg-1和CD206表达变化,并取其上清制成条件培养基,孵育Hela细胞,检测细胞凋亡和增殖情况。结果1)宫颈癌病变组织中METTL14 mRNA和蛋白表达降低(P<0.05),巨噬细胞M1型标志物IL-6和iNOS表达明显降低(P<0.05),而M2型标志物Arg-1和CD206表达明显升高(P<0.05)。2)巨噬细胞过表达METTL14后,IL-6和iNOS表达明显升高(P<0.05),而Arg-1和CD206表达明显降低(P<0.05),M1/M2比例升高;抑制METTL14表达后,M1型标志物降低(P<0.05),M2型标志物升高(P<0.05),M1/M2比例降低。3)巨噬细胞中转染OE-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被抑制(P<0.05);加入PI3K/AKT激动剂后,M1型标志物降低而M2型标记物升高(P<0.05),M1/M2比例降低;OE-METTL14可逆转此趋势。Sh-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被激活(P<0.05),加入PI3K/AKT抑制剂后,M1型标志物升高而M2型标记物降低(P<0.05),M1/M2比例升高;Sh-METTL14可逆转此趋势。4)取转染OE-METTL14慢病毒后的巨噬细胞上清培养Hela细胞,可见细胞凋亡明显增多(P<0.05),增殖明显减少(P<0.05)。Sh-METTL14组的Hela细胞则表现出细胞凋亡减少(P<0.05),增殖增多(P<0.05)。结论METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化可能有促进宫颈癌细胞凋亡,抑制增殖的作用。展开更多
N^(6)-methyladenosine(m^(6)A)modification,which is achieved by the METTL3/METTL14/WTAP methyltransferase complex,is the most abundant internal mRNA modification.Although recent evidence indicates that m^(6)A can regul...N^(6)-methyladenosine(m^(6)A)modification,which is achieved by the METTL3/METTL14/WTAP methyltransferase complex,is the most abundant internal mRNA modification.Although recent evidence indicates that m^(6)A can regulate neurodevelopment as well as synaptic function,the roles of m^(6)A modification in the cerebellum and related synaptic connections are not well established.Here,we report that Purkinje cell(PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to extensive PC degeneration and apoptotic cell death.Loss of Wtap also causes the aberrant degradation of multiple PC synapses.WTAP depletion leads to decreased expression levels of METTL3/14 and reduced m^(6)A methylation in PCs.Moreover,the expression of GFAP and NF-L in the degenerating cerebellum is increased,suggesting severe neuronal injuries.In conclusion,this study demonstrates the critical role of WTAP-mediated m^(6)A modification in cerebellar PCs,thus providing unique insights related to neurodegenerative disorders.展开更多
基金supported by the National Institute of Health to C.H.(Nos.HG008935,ES030546,and R01ES030546)the National Natural Science Foundation of China to J.L.(No.32170595)+1 种基金the Beijing Nova Program to JL.(No.Z211100002121011)the Center for Life Sciences(CLS),the School of Life Sciences(SLS)of Peking University,the SLS-Qidong Innovation Fund,and the Li Ge Zhao Ning Life Science Junior Research Fellowship.C.H.is an investigator of the Howard Hughes Medical Institute.
文摘METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell(mESC).While comparable global hypo-methylation in RNA m^(6)A was observed in Mettl3 or Mettl14 knockout mESCs,respectively.Mettl14 knockout led to a globally decreased nascent RNA synthesis,whereas Mettl3 depletion resulted in transcription upregulation,suggesting that METTL14 might possess an mA-independent role in gene regulation.We found that METTL14 colocalizes with the repressive H3K27me3 modification.Mechanistically,METTL14,but not METTL3,binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3.Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression.The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs.This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m^(6)A,and critically impacts transcriptional regulation,stemness maintenance,and differentiation ofmESCs.
文摘目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6、iNO、Arg-1和CD206表达变化以及PI3K/AKT/GSK3β/β-catenin信号通路相关蛋白表达情况。随后加入PI3K/AKT/GSK3β/β-catenin信号通路激动剂和抑制剂,检测过表达或抑制METTL14后,巨噬细胞IL-6、iNO、Arg-1和CD206表达变化,并取其上清制成条件培养基,孵育Hela细胞,检测细胞凋亡和增殖情况。结果1)宫颈癌病变组织中METTL14 mRNA和蛋白表达降低(P<0.05),巨噬细胞M1型标志物IL-6和iNOS表达明显降低(P<0.05),而M2型标志物Arg-1和CD206表达明显升高(P<0.05)。2)巨噬细胞过表达METTL14后,IL-6和iNOS表达明显升高(P<0.05),而Arg-1和CD206表达明显降低(P<0.05),M1/M2比例升高;抑制METTL14表达后,M1型标志物降低(P<0.05),M2型标志物升高(P<0.05),M1/M2比例降低。3)巨噬细胞中转染OE-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被抑制(P<0.05);加入PI3K/AKT激动剂后,M1型标志物降低而M2型标记物升高(P<0.05),M1/M2比例降低;OE-METTL14可逆转此趋势。Sh-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被激活(P<0.05),加入PI3K/AKT抑制剂后,M1型标志物升高而M2型标记物降低(P<0.05),M1/M2比例升高;Sh-METTL14可逆转此趋势。4)取转染OE-METTL14慢病毒后的巨噬细胞上清培养Hela细胞,可见细胞凋亡明显增多(P<0.05),增殖明显减少(P<0.05)。Sh-METTL14组的Hela细胞则表现出细胞凋亡减少(P<0.05),增殖增多(P<0.05)。结论METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化可能有促进宫颈癌细胞凋亡,抑制增殖的作用。
基金supported by the National Natural Science Foundation of China(82121003,81970841,and 81790643)the Department of Science and Technology of Sichuan Province(2021YFS0386,2021YFS0369,20ZYD038,20ZYD037,2020JDZH0026,2021JDZH0022)+2 种基金the CAMS Innovation Fund for Medical Sciences(2019-12M-5-032)Huanhua Distingished Scholar grantthe Department of Chengdu Science and Technology(2021-YF05-01316-SN)。
文摘N^(6)-methyladenosine(m^(6)A)modification,which is achieved by the METTL3/METTL14/WTAP methyltransferase complex,is the most abundant internal mRNA modification.Although recent evidence indicates that m^(6)A can regulate neurodevelopment as well as synaptic function,the roles of m^(6)A modification in the cerebellum and related synaptic connections are not well established.Here,we report that Purkinje cell(PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to extensive PC degeneration and apoptotic cell death.Loss of Wtap also causes the aberrant degradation of multiple PC synapses.WTAP depletion leads to decreased expression levels of METTL3/14 and reduced m^(6)A methylation in PCs.Moreover,the expression of GFAP and NF-L in the degenerating cerebellum is increased,suggesting severe neuronal injuries.In conclusion,this study demonstrates the critical role of WTAP-mediated m^(6)A modification in cerebellar PCs,thus providing unique insights related to neurodegenerative disorders.
