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

项目摘要 配子发生和早期胚胎发育的复杂事件需要严格的调控，以确保 健康的生殖和健康的后代。为了实现这种调节，同时保持必要的可塑性， 对于胚胎发生，生殖细胞严重依赖于基因表达的RNA水平调节。RNA调节在 在生殖细胞发育和维持中的许多重要作用，包括生殖细胞的维持- 特异性基因程序，保护种系基因组免受外来或破坏性序列的影响，以及 翻译抑制和储存的母体转录必要的早期胚胎发育之前， 合子转录的开始不同的RNA结合蛋白和小RNA通路网络控制着 这种至关重要的RNA调控，其中许多具有重叠或冗余的作用，以确保适当的调控 微调作用于RNA的腺苷脱氨酶（ADAR）是RNA结合蛋白，其可以影响RNA的表达。 通过与RNA结合或通过催化腺苷脱氨为肌苷， 称为A到I RNA编辑。ADAR存在于所有动物中，并已被证明在以下方面发挥重要作用： 发育、先天免疫和肿瘤发生。虽然ADAR在生殖细胞中的作用还没有被证实， 探索，我自己的初步数据表明ADAR在生殖系中高度表达，可以影响 数以百计的生殖系转录本。此外，其他实验室以前的工作表明，ADAR沿着 小RNA通路来执行正常繁殖所必需的功能。因此，我假设 ADAR在生殖系中发生的广泛RNA调控中发挥作用。我的初步 研究ADARs对生殖系转录物影响的实验表明，ADARs在以下方面发挥作用： 调节核糖体生物发生。此外，我发现许多生殖系编辑的转录本是已知的， 母体转录本在整个种系中受到翻译抑制，并储存以加载到 胚胎基于这些原因，我假设ADAR在翻译抑制中起作用， 在配子发生过程中贯穿整个生殖系。为了解决这个假设，我将扩展我的 研究ADAR对生殖系转录物的影响以及与其他生殖系RNA的相互作用 监管机构（目标1）。我将评估ADAR对生殖细胞核糖体生物发生和翻译的影响。 活动（目标2）。最后，我将研究阿达尔编辑对母系加载的转录本和早期转录本的影响。 胚胎RNA群体（Aim 3）。通过这些实验，我将了解 生殖系转录物上的ADAR，以及对它们在生殖细胞发育中的作用的更大理解， 维持，早期胚胎发育和一般生殖。这项工作将有助于我们 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.