Epitranscriptomic control of mRNA and noncoding RNAs in spermatogenesis

精子发生是一个精心安排的过程，在这个过程中精原干细胞分化为 精子细胞和最终可活动的精子。这个过程需要一系列表观遗传变化的改变， 染色质重组、mRNA3‘非编码区长度的动态变化和时间调节模式 翻译。新出现的证据表明，表位编码组中特定阶段的精确变化是 也是正常精子发生所必需的。例如，精母细胞特有的“阅读器”耗尽， N6-甲基腺苷的“写入者”或“擦除者”，N6-甲基腺苷是一种影响长非编码RNA的修饰核苷酸 (LncRNA)的功能和mRNA的稳定性，翻译和剪接，都与阶段特异性的停滞有关 精子发生。更多的数据表明，精子发生也受到N6，2‘-O- 二甲基腺苷(M6Am)，一种修饰的腺苷，仅在第一个转录的核苷酸上发现 某些mRNA的位置。基于这些研究，很明显表位转录修饰是 精子发生所必需的。然而，m6A和m6Am调控精子发生的机制 目前仍不清楚。为了破译表位转录组在精子发生中的作用，特定的目的是 本研究的主要内容是：(1)对精子发生过程中的m6A进行细胞类型定位和定量定位。我们 将开发新的方法来选择性地定位动物的M6A，并确定M6A控制的动态变化 MRNA3‘非编码区长度。这些方法将揭示精子发生和发育过程中m6A水平的动态变化 如果m6A功能参与了3‘非编码区长度动态的协调，这是精子发生的特征。 (2)确定m6A在精子发生过程中对表观基因组的调控作用。M6A通常富含 它可以影响它们诱导表观遗传基因沉默的能力。我们将鉴定染色质- 相关的包含m6A的lncRNA，并确定m6A如何影响表观遗传动力学 精子发生。(3)探讨YTHDC2和m6Am对精子发生的调控作用。YTHDC2是必需的 通过结合和调节m6A mRNAs来促进减数分裂进程。然而，YTHDC2表现出相对较弱的表现 与M6A结合。我们发现YTHDC2与m6Am有高亲和力结合我们将在M6Am期间绘制地图 精子发生，并通过耗尽其生物合成的甲基转移酶来确定m6Am的功能。我们会 还要确定YTHDC2的功能是调节m6Am mRNAs的稳定性或翻译 精子发生。总之，这些实验将揭示细胞类型的特定动力学 并将揭示这些表观转录修饰是如何影响表观遗传状态的 精子发生过程中的稳定性、信使核糖核酸3‘非编码区加工和信使核糖核酸的翻译。

Spermatogenesis is a carefully orchestrated process in which spermatogonial stem cells differentiate into spermatids and eventually motile sperm. This process requires a series of changes in epigenetic changes, chromatin reorganization, dynamic changes in mRNA 3’UTR length, and temporally regulated patterns of translation. Emerging evidence suggests that precise stage-specific alterations in the epitranscriptome are also required for proper spermatogenesis. For example, spermatocyte-specific depletion of “readers,” “writers,” or “erasers,” of N6-methyladenosine, a modified nucleotide that impacts long noncoding RNA (lncRNA) function and mRNA stability, translation, and splicing, are all associated with stage-specific arrests in spermatogenesis. Additional data suggests that spermatogenesis is also affected by N6, 2’-O- dimethyladenosine (m6Am), a modified adenosine that is found exclusively at the first transcribed nucleotide position of certain mRNAs. Based on these studies, it is clear that epitranscriptomic modifications are required for spermatogenesis. However the mechanisms by which m6A and m6Am regulate spermatogenesis remain unclear. In order to decipher the role of the epitranscriptome in spermatogenesis, the specific aims of this project are: (1) To map m6A in a cell-type specific and quantitative manner during spermatogenesis. We will develop new methods to selectively map m6A in animals, and determine if dynamic changes in m6A control mRNA 3’UTR length. These methods will reveal the dynamics of m6A levels throughout spermatogenesis and if m6A function is involved in orchestrating 3’UTR length dynamics that is characteristic of spermatogenesis. (2) To determine the role of m6A in controlling the epigenome during spermatogenesis. m6A is often enriched in lncRNAs, and it can affect their ability to induce epigenetic gene silencing. We will identify chromatin- associated lncRNAs that contain m6A and determine how m6A affects epigenetic dynamics during spermatogenesis. (3) To determine how YTHDC2 and m6Am regulate spermatogenesis. YTHDC2 is required for meiotic progression by binding and regulating m6A mRNAs. However, YTHDC2 shows relatively weak binding to m6A. We find that YTHDC2 shows high-affinity binding to m6Am. We will map m6Am during spermatogenesis, and determine the function of m6Am by depleting its biosynthetic methyltransferase. We will also determine if the function of YTHDC2 is to regulate the stability or translation of m6Am mRNAs during spermatogenesis. Together, these experiments will reveal the cell-type specific dynamics of the epitranscriptome and will reveal how these epitranscriptomic modifications affect epigenetic states, mRNA stability, mRNA 3’UTR processing and mRNA translation during spermatogenesis.