Investigating the Roles of ADARs and A-to-I RNA Editing in Germline RNA Regulation

项目总结 配子发生和早期胚胎发育的复杂事件需要严格的监管，以确保 正常繁殖和健康的后代。要在保持必要的可塑性的同时实现这一调节 对于胚胎发育，生殖细胞在很大程度上依赖于RNA水平的基因表达调控。RNA调控发挥着重要作用 生殖细胞发育和维持中的一些重要作用，包括生殖细胞的维持- 特定的基因程序，保护生殖系基因组免受外来或破坏性序列的影响，以及 翻译抑制和储存早期胚胎发育所需的母体转录本 合子转录的开始。一个由RNA结合蛋白和小RNA通路组成的多样化网络 这一关键的RNA调控，其中许多具有重叠或多余的作用，以确保适当的调控 微调。作用于RNA的腺苷脱氨酶(ADAR)是一种RNA结合蛋白，可以影响 转录物通过与RNA结合或催化腺苷脱氨为肌苷的细胞命运， 称为A-to-I RNA编辑。ADAR存在于所有动物中，并已被证明在 发育、先天免疫和肿瘤发生。虽然ADAR在生殖细胞中的作用尚未被 我自己的初步数据表明，ADAR在生殖系中高度表达，并可以影响 数百份生殖系成绩单。此外，其他实验室之前的工作表明，ADAR也会发挥作用 具有执行正常繁殖所必需的功能的小RNA途径。因此，我假设 ADARS在生殖系中发生的广泛的RNA调节中发挥作用。我的初选 研究ADAR对生殖系转录本影响的实验表明，ADAR在 调节核糖体的生物发生。此外，我发现许多生殖系编辑的记录都是已知的 母系转录本，在整个生殖系中被翻译抑制，并存储以加载到 胚胎。出于这些原因，我假设ADAR在发生的翻译抑制中发挥了作用 在配子发生过程中贯穿整个生殖系。为了回答这一假设，我将扩展我的 ADARs对生殖系转录本的影响以及与其他生殖系RNA相互作用的研究 监管机构(目标1)。我将评估ADAR对生殖系核糖体生物发生和翻译的影响 活动(目标2)。最后，我将调查ADAR编辑对母亲加载的成绩单和早期的影响 胚胎RNA种群(目标3)。通过这些实验，我将对 关于生殖系转录本的ADARS，以及对它们在生殖细胞发育和 通常是维持、早期胚胎发育和繁殖。这项工作将为我们的 了解促进成功和健康生殖的复杂过程。

PROJECT SUMMARY The complex events of gametogenesis and early embryonic development require tight regulation to ensure proper reproduction and healthy offspring. To achieve this regulation while maintaining the plasticity necessary for embryogenesis, germ cells rely heavily on RNA-level regulation of gene expression. RNA regulation plays a number of important roles in germ cell development and maintenance, including maintenance of germ cell- specific gene programs, protection of the germline genome from foreign or damaging sequences, and translational repression and storage of maternal transcripts necessary for early embryonic development prior to the onset of zygotic transcription. A diverse network of RNA binding proteins and small RNA pathways govern this crucial RNA regulation, many of which have overlapping or redundant roles to ensure proper regulatory fine-tuning. Adenosine DeAminases that act on RNA (ADARs), are RNA binding proteins that can influence the cellular fate of transcripts either by binding to RNA or by catalyzing the deamination of Adenosine to Inosine, known as A-to-I RNA editing. ADARs are present in all animals and have been shown to play important roles in development, innate immunity, and oncogenesis. While the roles of ADARs in germ cells has not been explored, my own preliminary data suggests ADARs are highly expressed in the germline and can influence hundreds of germline transcripts. Additionally, previous work from other labs suggests that ADARs act along with small RNA pathways to perform functions necessary for proper reproduction. As such, I hypothesize that ADARs play a role in the extensive RNA regulation that takes place in the germline. My preliminary experiments investigating the effects of ADARs on germline transcripts suggest that ADARs play a role on regulating ribosome biogenesis. Additionally, I have found that many germline-edited transcripts are known maternal transcripts, which are translationally repressed throughout the germline and stored for loading into the embryo. For these reasons, I hypothesize that ADARs play a role in translational repression that occurs throughout the germline during gametogenesis. To address this hypothesis, I will expand upon my investigation of the effects of ADARs on germline transcripts as well as interactions with other germline RNA regulators (Aim 1). I will assess the impact of ADARs on germline ribosome biogenesis and translational activity (Aim 2). Finally, I will investigate the impact of ADAR editing on maternally loaded transcripts and early embryonic RNA populations (Aim 3). Through these experiments, I will gain an understanding of the effects of ADARs on germline transcripts, and a bigger-picture understanding of their role in germ cell development and maintenance, early embryonic development, and reproduction in general. This work will contribute to our understanding of the complex processes that facilitate successful and healthy reproduction.