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-κB 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. 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) .

作为N6-甲基腺苷（m6A）修饰写入器（writer）的METTL3-METTL14复合物，在众多生物学过程中发挥关键调控作用。既往研究显示，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）复合物另一核心组分的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。当与Oct4、Sox2、Klf4及c-Myc（OSKM）协同作用时，Mettl14可在重编程后期短暂上调非重编程细胞中衰老相关分泌表型（SASP）基因的表达。过表达Mettl4或其突变体的重编程中间体细胞的条件培养基，可增强重编程效率；作为SASP组分之一的IL-6亦具有类似效果。相应地，使用衰老消融剂或核因子κB（NF-κB）抑制剂阻断SASP通路，会削弱Mettl14对细胞重编程的促进作用。本研究揭示了Mettl14不依赖m6A的全新生物学功能，并为体外衰老与重编程之间的相互调控关系提供了新的研究视角。本实验所用细胞为源自携带tetO-OSKM转基因及Oct4-GFP/Rosa26-M2rtTA遗传元件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"。将过表达或未过表达Obox1的重编程细胞以1μg/ml浓度的强力霉素诱导培养3天，随后收集细胞并开展RNA测序（RNA-seq）实验。