Mouse models for live-cell imaging of endogenous Evf2 lncRNA

摘要 长的非编码RNA(LncRNAs)在不同的细胞过程中发挥着关键作用， 包括转录调控和转录后调控。EVF2超长时间服务 LncRNA是一种转录调控的lncRNA，由Dlx5/6超微结构转录而来 发育中的前脑的增强子(UCE)。与参与印迹的lncRNA不同， 单向调节基因表达，EVF2正负向调节靶点 基因。EVF2与87种蛋白质和对照形成核糖核蛋白复合体(EVF2-RNP) 影响神经元间多样性和癫痫发作的基因调控网络的表达 易感性，通过涉及转录因子招募的机制， 以及表观遗传修饰和染色体拓扑的控制。 研究EVF2的亚核定位是了解其功能的关键 功能。EVF2在每个细胞核上形成一到两个RNA云。EVF2-RNP蛋白的一个亚集 定位于与EVF2 RNA云共同定位的焦点(蛋白池)。共同本地化 含有Dlx5/6UCE和EVF2调控基因的EVF2-RNP揭示了重要的 有关EVF2依赖的基因调控的机制信息。有趣的是，EVF2并非如此 总是保留在其表达部位(Dlx5/6UCE)，但可以与EVF2- 独立于UCE的受调控靶点，表明EVF2与 染色体是动态的。 EVF2-RNP在单细胞水平的可视化提供了基本信息 以了解时空分辨率下的EVF2调控机制。然而， 利用固定细胞的成像实验未能揭示关于动态的重要信息 流程。在这项提案中，我们将利用RNA活细胞成像的最新进展 EVF2在活细胞中的可视化。我们将为这项研究制作小鼠模型 EVF2云形成、EVF2云动力学和EVF2-RNP形成。这项研究将 回答关键问题以促进对机制的了解 LncRNAs的转录调控。

Abstract Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes, including transcriptional and post-transcriptional regulation. The Evf2 ultraconserved lncRNA is a transcription regulating lncRNA transcribed from the Dlx5/6 ultraconserved enhancer (UCE) in the developing forebrain. Unlike lncRNAs involved in imprinting, that regulate gene expression in one direction, Evf2 positively and negatively regulates target genes. Evf2 forms a ribonucleoprotein complex (Evf2-RNP) with 87 proteins and controls the expression of a gene regulatory network that affects interneuron diversity and seizure susceptibility, through mechanisms that involve the recruitment of transcription factors, and the control of epigenetic modifications and chromosome topology. The study of the subnuclear localization of Evf2 is key for understanding its function. Evf2 forms one or two RNA clouds per nucleus. A subset of Evf2-RNP proteins localize in foci (protein pools) that co-localize with the Evf2 RNA cloud. Co-localization of the Evf2-RNP with the Dlx5/6 UCE and Evf2-regulated genes, reveals important mechanistic information about Evf2-dependent gene regulation. Intriguingly, Evf2 is not always retained at its site of expression (the Dlx5/6 UCE) but can interact with Evf2- regulated targets independent of the UCE, suggesting that interactions of Evf2 with the chromosome are dynamic. Visualization of the Evf2-RNP at the single-cell level provides essential information for understanding Evf2 regulatory mechanisms at spatiotemporal resolution. However, imaging experiments utilizing fixed cells fail to reveal important information about dynamic processes. In this proposal, we will utilize recent advances in live-cell imaging of RNAs for the visualization of Evf2 in living cells. We will generate mouse models for the study of Evf2 cloud formation, Evf2 cloud dynamics, and Evf2-RNP formation. This study will answer critical questions for the advancement of knowledge about mechanisms of transcriptional regulation by lncRNAs.