ERK-mediated regulation of non-coding RNAs during development and disease

项目总结 通过精子和卵母细胞的融合成功繁殖对于物种的永久存在是必不可少的。 在人类女性中，卵母细胞在出生时完成减数分裂i，并进入长时间的减数分裂ii停滞期，直到 在青春期开始减数分裂成熟。因为在此期间卵母细胞处于静止和停滞状态， RNAs在受阻前被加载到发育中的卵母细胞中，这些RNAs是早期发育的关键 胚胎发育。调节产生和保护(免受退化)的机制 长时间减数分裂停滞过程中的母体RNA以及调节这些RNA降解的机制 胚胎中的RNA仍然是一个活跃的研究领域。我们在线虫上的工作和在哺乳动物上的工作 过去几年的模型将目光投向了母体转录组的调控，转录组决定了卵母细胞的发育 质量和影响后代发育。具体地说，我们发现RAS/ERK增长之间存在直接联系 因子信号转导与小RNA生物发生因子Dicer1、DROSHA和DIS3(RNA外体成分) 它调节不同的小非编码RNA群体，从而调节母体转录组和 蛋白质组。我们提出了一个模型，其中ERK介导的Dicer1(和随后的精氨酸)的磷酸化 Dicer1)的甲基化，DROSHA和DIS3的磷酸化导致调节电路微调 在特定的亚群中产生小的非编码RNA，并调节母体和合子转录组 和蛋白质组。我们在体内研究了这种模型在卵母细胞发育和卵母细胞到胚胎的转变过程中的作用。 使用实时成像、下一代测序、单卵母细胞测序、质谱分析 蛋白质组学方法、CRISPR Cas9基因组编辑和细胞生物学检测。我们发现Dicer1，DROSHA 和Dis3在哺乳动物中也是磷酸化的。此外，我们还鉴定了Dicer1的精氨酸甲基化 在哺乳动物细胞培养系统中与磷酸化事件相邻。鉴于它们在RNA中的保守作用 生物学、生殖及其与癌症发生和发展相关的异常，我们期待着这项工作 与人类生物学有直接关系。

PROJECT SUMMARY Successful reproduction through the fusion of the sperm and oocyte is essential for the perpetuation of species. In human females, oocytes complete meiosis I at birth, and enter a long period of meiotic II arrest until onset of meiotic maturation at puberty. Because the oocytes are quiescent and arrested during this period, RNAs are loaded into the developing oocytes prior to the arrest and these RNAs are critical for early embryonic development. Mechanisms that regulate generation and protection (from degradation) of maternal RNAs during the long meiotic arrest as well as mechanisms that regulate the degradation of these RNAs in the embryo remain an active area of investigation. Our work in C. elegans and work from mammalian models in the past few years turned the light on regulation of the maternal transcriptome which dictates oocyte quality and impacts progeny development. Specifically, we uncovered a direct link between RAS/ERK growth factor signaling and the small RNA biogenesis factors Dicer1, Drosha and DIS3 (an RNA exosomal component) which regulates distinct populations of small non-coding RNAs and thus the maternal transcriptome and proteome. We propose a model wherein ERK-mediated phosphorylation of Dicer1 (and a subsequent arginine methylation of Dicer1), phosphorylation of Drosha and DIS3 results in a regulatory circuit that fine tunes the generation of small non-coding RNAs in specific subsets and regulates the maternal and zygotic transcriptome and proteome. We investigate this model in vivo during oocyte development and oocyte-to-embryo transition using a combination of live imaging, next generation sequencing, single oocyte sequencing, mass spectrometric and proteomic methods, CRISPR Cas9 genome editing and cell biological assays. We find that Dicer1, Drosha and Dis3 are phosphorylated in mammals as well. Additionally, we identified arginine methylation of Dicer1 adjacent to the phosphorylation event in mammalian cell culture system. Given their conserved role in RNA biology, reproduction and their aberrations associated with cancer onset and progression, we expect this work to have direct relevance to human biology.