Epitranscriptomic regulation of spermatogenesis and male fertility

项目摘要 最近，对mRNAs的可逆化学修饰被认为是一种主要的调控 基因表达的机制。在迄今确认的100种RAN化学修饰中，N6- 甲基腺苷(M6A)是真核生物中含量最丰富的一种，每个mRNA3-5个m6A位点 抄本。我们最近的研究(PNAS，2017,115：E325)证明了ALKBH5作为一种 M6A橡皮擦，以及M6A作为精母细胞核选择性剪接的信号 圆形精子细胞，以及细长和细长精子细胞的细胞质中的降解。这个 正确的表位转录调控(例如，m6A)对于成功是必不可少的科学前提 精子发生和男性生育能力促使我们着手进行研究，旨在了解 在发育中的雄性生殖细胞中，m6a水平是如何被控制的？这种特殊的化学物质起什么作用？ MRNA的修饰在精子发生的调控中起着重要作用。基于我们发布的和 初步数据，我们假设ALKBH4，一个ALKBH5的同源物，调节m6A的mRNA水平在 生精细胞作为ALKBH5的一种新的擦除剂或辅助因子。为了检验这一假设，我们将 首先确定ALKBH4是作为一种新的RNA m6A去甲基酶还是作为一种辅助因子 ALKBH5采用体外生化分析(Aim1)。然后，将使用以下方法验证体外发现 小鼠体内基因敲除模型及ALKBH4的生理调节作用 精子发生也将被确定(AIM2)。拟议的项目将帮助我们深入了解 精子发生的表型转录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.