Epitranscriptomic regulation of spermatogenesis and male fertility

项目摘要 mRNA的可逆化学修饰最近被认为是一种主要的调控机制。 基因表达的机制。在迄今为止鉴定的>100种各种RAN化学修饰中，N6- 甲基腺苷（m6 A）是真核生物中最丰富的一种，每个mRNA约有3-5个m6 A位点 转录组我们最近的工作（PNAS，2017，115：E325）已经证明ALKBH 5作为一种蛋白质， m6 A擦除器，m6 A作为精母细胞核中选择性剪接的信号， 圆形精子细胞，以及在伸长和伸长精子细胞的细胞质中降解。的 科学前提是适当的表转录调控（例如，m6 a）是成功的关键 精子发生和男性生育力促使我们开始研究， m6 A水平在男性生殖细胞发育中是如何控制的，这种特定的化学物质在其中扮演什么角色？ mRNA的修饰在精子发生中起调节作用。根据我们发布的和 根据初步数据，我们假设ALKBH 4，ALKBH 5的同源物，调节mRNA m6 A水平， 生精细胞作为一种新的橡皮擦或ALKBH 5的辅助因子。为了验证这个假设，我们将 首先确定ALKBH 4是否作为一种新的RNA m6 A脱甲基酶或作为一种辅助因子， ALKBH 5使用体外生化分析（Aim 1）。然后将使用以下方法验证体外结果： 基因敲除小鼠体内模型和ALKBH 4在调节 还将测定精子发生（Aim 2）。拟议的项目将帮助我们了解 精子发生的表观调控和所获得的知识将有助于我们发现 精子质量差和男性不育的根本原因。

Project Summary Reversible chemical modifications of mRNAs have recently been recognized as a major regulatory mechanism of gene expression. Among >100 various RAN chemical modifications identified so far, N6- methyladenosine (m6A) represents the most abundant one with ~3-5 m6A sites per mRNA in eukaryotic transcriptomes. Our recent work (PNAS, 2017, 115:E325) has demonstrated that ALKBH5 acts as a m6A eraser, and m6A serves as a signal for alternative splicing in the nucleus of spermatocytes and round spermatids, and for degradation in the cytoplasm of elongating and elongated spermatids. The scientific premise that proper epitranscriptomic regulation (e.g., m6A) is essential for successful spermatogenesis and male fertility prompted us to embark on investigations aiming to understand how m6A levels are controlled in developing male germ cells and what role this specific chemical modification of mRNA plays in the regulation of spermatogenesis. Based upon our published and preliminary data, we hypothesize that ALKBH4, a homolog of ALKBH5, regulates mRNA m6A levels in spermatogenic cells either as a novel eraser or a co-factor of ALKBH5. To test this hypothesis, we will first determine whether ALKBH4 functions as a novel RNA m6A demethylase or as a co-factor for ALKBH5 using biochemical analyses in vitro (Aim1). The in vitro findings will then be validated using knockout mouse models in vivo and the physiological role of ALKBH4 in the regulation of spermatogenesis will also be determined (Aim2). The proposed project will help us gain insights into the epitranscriptomic regulation of spermatogenesis and the knowledge gained would help us discover the underlying causes of poor sperm quality and male infertility.