YTHDC2 regulates spermatogenesis through promoting the translation of N6-methyladenosine-modified RNA

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as an essential layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to promote the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the splicing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A along its consensus motif GGACU. YTHDC2 promotes translation of its targets, and associates with cellular fractions involved in translation initiation. Ythdc2 knockout mice are infertile and have significantly smaller testes compared to those of littermates. In the testes, Ythdc2 is temporally expressed as meiosis begins, and germ cells of Ythdc2 knockout mice do not develop past the spermatogonium stage. Thus, YTHDC2 is an m6A binding protein that plays essential roles in spermatogenesis. Overall design: We used CLIP-seq replicates and RIP-seq replicates to identify the binding targets and sites of YTHDC2. We used m6A-seq to identify changes in m6A and RNA expression in Ythdc2-/- mice

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是真核信使RNA（mRNA）中最常见的内部修饰形式。该修饰可被动态催化添加与移除，是mRNA代谢调控的关键层级，参与调控干细胞多能性、细胞分化及能量稳态等多种生物学过程。m6A可被选择性结合蛋白识别：YTHDF1与YTHDF3协同作用，促进携带m6A的mRNA的翻译；YTHDF2可加速靶mRNA的降解；YTHDC1则调控其靶标的剪接过程。作为YTH蛋白家族的最后一个成员，YTHDC2的生物学功能此前尚未明确。
本研究证实，YTHDC2可沿其保守基序GGACU选择性结合m6A。YTHDC2能够促进其靶标的翻译，并与参与翻译起始的细胞组分相结合。Ythdc2基因敲除小鼠表现为不育，且睾丸体积显著小于同窝仔鼠。在睾丸组织中，Ythdc2在减数分裂起始时呈时序性表达，而Ythdc2基因敲除小鼠的生殖细胞无法突破精原细胞阶段完成发育。综上，YTHDC2是一种m6A结合蛋白，在精子发生过程中发挥关键调控作用。
整体实验设计：本研究通过紫外交联免疫沉淀测序（CLIP-seq）重复实验与RNA免疫沉淀测序（RIP-seq）重复实验，鉴定YTHDC2的结合靶标与结合位点；并采用m6A测序（m6A-seq）分析Ythdc2基因敲除（Ythdc2-/-）小鼠体内m6A修饰水平与RNA表达的变化。