A novel RNA m6A modulator Zc3h13 plays an anchor role in facilitating nuclear RNA methylation and mouse embryonic stem cell self-renewal. Mus musculus

N6-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export and translation. Recent studies discovered m6A methyltransferases (?writer?), demethylases (?eraser?) and binding proteins (?reader?), which modulate m6A methylation. However, the precise m6A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc finger protein, plays an essential role in modulating m6A methylation on polyadenylated RNA in the nucleus. ZC3H13 exists in an evolutionary-conserved macromolecular complex containing WTAP, Virilizer and Hakai. We confirm the interaction among those proteins and demonstrate that knockdown of Zc3h13 in mouse embryonic stem cell (mESC) significantly decreases global m6A level on mRNA, mainly at 3? untranslated regions (3? UTR). Interestingly, fractionation assays show that upon Zc3h13 knockdown a great majority of WTAP, Virilizer and Hakai translocate to the cytoplasm and the nuclear presence of the methyltransferase Mettl3 and Mettl14 also decrease significantly. In contrast, knockdown of WTAP, Virilizer or Hakai does not change the nuclear localization of Zc3h13. This suggests that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays an essential role in anchoring WTAP, Virilizer and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal. Overall design: We examined N6-methyladenosine and mRNA profiles in scr and shZc3H13 mouse embryonic stem cells. MeRIP-seq were performed to examine N6-methyladenosine (m6A) on the transcrits in the cytoplasmic and nuclear fractions of control and Zc3h13 kd mESCs. RNA-seq were conducted to examine the gene expression profiles of Zc3h13 kd and control mESCs. 3, RIP-seq were carried out to identify Zc3h13 protein binding RNAs in mESCs.

N6-甲基腺嘌呤（N6-methyladenosine, m6A）是真核mRNA中一种丰度较高的修饰，可通过影响mRNA稳定性、剪接、输出与翻译过程调控mRNA的代谢动态。已有研究发现m6A甲基转移酶（writer）、去甲基化酶（eraser）以及结合蛋白（reader）可调控m6A甲基化修饰，但m6A的精准调控机制仍未完全阐明。本研究证实，锌指蛋白ZC3H13可对细胞核内多腺苷酸化RNA的m6A甲基化修饰发挥关键调控作用。ZC3H13存在于一个进化保守的大分子复合物中，该复合物包含WTAP、Virilizer与Hakai。我们验证了这些蛋白之间的相互作用，并发现小鼠胚胎干细胞（mouse embryonic stem cell, mESC）中Zc3h13敲低会显著降低mRNA整体的m6A修饰水平，且该降低主要发生在3'非翻译区（3' UTR）。有趣的是，细胞组分分离实验显示，Zc3h13敲低后，绝大多数WTAP、Virilizer与Hakai会转位至细胞质，且细胞核内甲基转移酶Mettl3与Mettl14的含量也显著降低。与之相反，敲低WTAP、Virilizer或Hakai并不会改变ZC3H13的核定位。这表明ZC3H13是Zc3h13-WTAP-Virilizer-Hakai复合物定位于细胞核的必要条件，而该复合物的核定位对RNA m6A甲基化修饰至关重要。最后，与敲低WTAP、Virilizer或Hakai一致，Zc3h13缺失会损害小鼠胚胎干细胞的自我更新能力并诱导其分化。综上，本研究结果表明，ZC3H13可通过将WTAP、Virilizer与Hakai锚定在细胞核内，从而促进RNA的m6A甲基化修饰并调控小鼠胚胎干细胞的自我更新。

整体实验设计：我们检测了阴性对照（scr）与Zc3h13敲低（shZc3H13）小鼠胚胎干细胞中的N6-甲基腺嘌呤与mRNA表达谱。通过甲基化RNA免疫沉淀测序（MeRIP-seq），我们检测了对照与Zc3h13敲低小鼠胚胎干细胞的细胞质与细胞核组分中转录本的N6-甲基腺嘌呤（m6A）修饰水平；通过RNA测序（RNA-seq）分析了Zc3h13敲低与对照小鼠胚胎干细胞的基因表达谱；通过3'端RNA免疫沉淀测序（3' RIP-seq）鉴定了小鼠胚胎干细胞中ZC3H13蛋白结合的RNA。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于Zc3h13在小鼠胚胎干细胞中调控RNA m6A甲基化的关键作用。研究发现Zc3h13与WTAP、Virilizer、Hakai形成复合物，锚定于细胞核以促进m6A修饰，敲低Zc3h13会降低全局m6A水平并触发干细胞分化。实验采用MeRIP-seq、RNA-seq和RIP-seq技术，全面分析m6A修饰、基因表达和蛋白质结合，揭示了Zc3h13在干细胞自我更新中的分子机制。