Regulation of m6A transcripts by the 3'->5' RNA helicase YTHDC2 is essential for a successful meiotic program in the mammalian germline

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. In mutant males, mitotic spermatogonia fail to differentiate into meiotic spermatocytes. Analysis identifies transcripts upregulated in the Yhtdc2 mutant testes to be those that are expressed in purified spermatogonia, while high-throughput mapping of the m6A transcriptome in the mouse male germline demonstrates the upregulated transcripts to be those that are enriched in m6A marks. Our biochemical studies indicate that YTHDC2 is an RNAinduced ATPase that fuels its 3'->5' RNA helicase activity. Interestingly, we find an RNA-independent interaction between the 5'->3' exoribonuclease XRN1 and the Ankyrin repeat of YTHDC2 that is wedged in between the two RecA helicase domains. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts that is essential for successful meiosis. RNA sequencing was used to identify transcript abundance differences between Ythdc2 mutant and wt mice. The distribution of m6A in testicular transcripts was analyzed by m6A RNA-Seq. RNA Bind-N-Seq analysis was performed to determine the YTH domain bound sequences.N6-甲基腺嘌呤（N6-methyladenosine，m6A）是一类至关重要的内部RNA修饰，在绝大多数生物中对基因表达调控发挥核心功能。携带YTH结构域（YTH domain）的蛋白能够识别m6A修饰标记，并作为分子功能介导因子参与RNA剪接、mRNA降解及翻译调控等关键生理过程。本研究证实，含YTH结构域蛋白2（YTHDC2）是一种m6A阅读蛋白，对小鼠的雌雄生育能力不可或缺。在Ythdc2突变的雄性小鼠体内，有丝分裂精原细胞无法分化为减数分裂精母细胞。转录组分析显示，Ythdc2突变小鼠睾丸中上调的转录本，正是纯化精原细胞中特异性表达的转录本；而对小鼠雄性生殖细胞系的m6A转录组进行高通量图谱绘制后发现，上述上调的转录本恰恰是富集m6A修饰的转录本。生化实验表明，YTHDC2是一种RNA诱导型ATP酶（ATPase），可为其3'→5' RNA解旋酶活性提供能量。有趣的是，本研究还发现，5'→3'外切核糖核酸酶XRN1（XRN1）与YTHDC2的锚蛋白重复序列（Ankyrin repeat）之间存在不依赖RNA的相互作用，该锚蛋白重复序列恰好嵌入两个RecA解旋酶结构域（RecA helicase domains）之间。本研究揭示了YTHDC2在调控m6A修饰的生殖细胞系转录本水平中发挥的关键作用，这一作用对顺利完成减数分裂至关重要。研究人员通过RNA测序（RNA sequencing）技术鉴定了Ythdc2突变型与野生型（wt）小鼠之间的转录本丰度差异；通过m6A RNA测序（m6A RNA-Seq）分析了睾丸转录本中的m6A分布特征；并通过RNA结合测序（RNA Bind-N-Seq）分析确定了YTH结构域的结合序列。