Mettl14-driven senescence-associated secretory phenotype (SASP) facilitates somatic reprogramming

The METTL3-METTL14 complex, as the "writer" of N6-methyladenosine (m6A), plays an important role in many biological processes. Previous studies have shown that overexpression of Mettl3 can increase the level of m6A and promotes somatic cell reprogramming. Here, we demonstrate that Mettl14, another component of the methyltransferase (MTase) complex, can significantly enhance the generation of induced pluripotent stem cells (iPSCs) in m6A independent manner. Cooperating with Oct4, Sox2, Klf4 and c-Myc (OSKM), Mettl14 transiently increased the senescence-associated secretory phenotype (SASP) gene expression in the non-reprogramming cells at the late reprogramming stage. The conditional medium in reprogramming intermediates overexpressing Mettl4 or its mutant could enhanced the reprogramming, so do IL-6, a component of SASP. Corespondingly, blocking of SASP using senolytic agent or NF-?B inhibitor impairs the effect of Mettl14 on reprogramming. . Our work highlights the m6A independent function of Mettl14 and provides new insight into the interplay between senescence and reprogramming in vitro. Overall design: The reprogrammable MEFs derived from the transgenic mice carrying the tetO-OSKM transgene and Oct4-GFP/Rosa26-M2rtTA were used in the experiments. The reprogrammable MEFs that were not induced with doxycycline were designated as “MEF” . The reprogrammable cells with or without Obox1's overexpression were induced with doxycycline (1ug/ml) for 3 days. The cells were harvested and performed RNA sequencing (RNA-seq) .

METTL3-METTL14复合物作为N6-甲基腺苷（m6A）的写入因子，广泛参与诸多生物学过程。既往研究表明，过表达Mettl3可提升细胞内m6A水平，并促进体细胞重编程。本研究证实，作为甲基转移酶（MTase）复合物另一亚基的Mettl14，能够以不依赖m6A的方式显著增强诱导多能干细胞（iPSCs）的生成效率。在与Oct4、Sox2、Klf4及c-Myc（OSKM）协同作用时，Mettl14可在重编程后期暂时性上调非重编程细胞中衰老相关分泌表型（SASP）相关基因的表达。过表达Mettl4及其突变体的重编程中间细胞的条件培养基，可提升重编程效率；作为SASP组分的IL-6亦具有相同效果。相应地，使用衰老清除剂或NF-κB抑制剂阻断SASP通路，会削弱Mettl14对重编程的促进作用。本研究揭示了Mettl14不依赖m6A的新功能，并为体外条件下衰老与重编程之间的相互调控关系提供了全新视角。实验整体设计：本实验采用源自携带tetO-OSKM转基因与Oct4-GFP/Rosa26-M2rtTA的转基因小鼠的可重编程小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts，MEFs）。未用强力霉素诱导的可重编程MEFs被命名为"MEF"组。分别转染或不转染Obox1过表达载体的可重编程细胞，均用1μg/ml强力霉素诱导3天。收集细胞并进行RNA测序（RNA-seq）。