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

项目摘要 通过精子和卵母细胞的融合成功繁殖对于物种的延续是必不可少的。 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。由于卵母细胞在此期间处于静止和停滞状态， RNA在停滞前被加载到发育中的卵母细胞中，这些RNA对于早期发育至关重要。 胚胎发育调节生物质的产生和保护（免于退化）的机制 母体RNA在长减数分裂停滞期间以及调节这些降解的机制， 胚胎中的RNA仍然是一个活跃的研究领域。我们在C的工作哺乳动物的研究 在过去几年中，一些模型揭示了决定卵母细胞的母体转录组的调控 质量和影响后代发育。具体来说，我们发现RAS/ERK的增长与 因子信号传导和小RNA生物合成因子Dicer 1、Drosha和DIS 3（RNA外泌体组分） 其调节小的非编码RNA的不同群体，从而调节母体转录组， 蛋白质组我们提出了一个模型，其中ERK介导的Dicer 1磷酸化（和随后的精氨酸 Dicer 1的甲基化）、Drosha和DIS 3的磷酸化导致了一个调节回路， 在特定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。我们在卵母细胞发育和卵母细胞向胚胎转化过程中研究了这种模型 使用实时成像、下一代测序、单个卵母细胞测序、质谱 和蛋白质组学方法、CRISPR Cas9基因组编辑和细胞生物学测定。我们发现Dicer 1，Drosha 和Dis 3在哺乳动物中也被磷酸化。此外，我们鉴定了Dicer 1的精氨酸甲基化， 与哺乳动物细胞培养系统中的磷酸化事件相邻。鉴于它们在RNA中的保守作用 生物学，生殖及其与癌症发病和进展相关的畸变，我们期待这项工作 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.