Epitranscriptomic control of mRNA and noncoding RNAs in spermatogenesis

精子发生是一个精心安排的过程，其中精原干细胞分化为 精子细胞和最终活动的精子。这个过程需要一系列表观遗传变化， 染色质重组，mRNA 3 'UTR长度的动态变化，以及 翻译.新出现的证据表明，在epitranscriptome中精确的阶段特异性改变是 这也是正常精子发生所必需的。例如，精母细胞特异性的“读者”耗竭， N6-甲基腺苷的“写入器”或“擦除器”，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，都与阶段特异性逮捕， 精子发生其他数据表明，精子发生也受到N6，2 '-O-的影响。 二甲基腺苷（m6 Am），一种仅在第一个转录核苷酸处发现的修饰腺苷 某些mRNA的位置。基于这些研究，很明显， 精子生成所必需的。然而，m6 A和m6 Am调节精子发生的机制 仍然不清楚。为了解释表转录组在精子发生中的作用， 本研究的主要目的是：（1）对精子发生过程中的m6 A进行细胞类型特异性的定量定位。我们 将开发新的方法来选择性地绘制动物中的m6 A，并确定m6 A控制的动态变化是否 mRNA 3 'UTR长度。这些方法将揭示整个精子发生过程中m6 A水平的动态变化， m6 A功能是否参与了精子发生特征性的3 'UTR长度动态的协调。 (2)探讨m6 A在精子发生过程中对表观基因组的调控作用。m6 A通常富含 lncRNA，并且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lncRNA包含m6 A，并决定m6 A如何影响表观遗传动力学， 精子发生(3)确定YTHDC 2和m6 Am如何调节精子发生。需要YTHDC 2 通过结合和调节m6 A mRNA来促进减数分裂进程。然而，YTHDC 2显示相对较弱 与m6 a结合。我们发现YTHDC 2与m6 Am表现出高亲和力结合。我们将绘制m6 am， 精子发生，并通过消耗其生物合成甲基转移酶来确定m6 Am的功能。我们将 还确定YTHDC 2的功能是否是调节m6 Am mRNA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， epitranscriptome和将揭示这些epitranscriptomic修改如何影响表观遗传状态，mRNA 精子发生过程中的稳定性、mRNA 3 'UTR加工和mRNA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.