Mouse models for live-cell imaging of endogenous Evf2 lncRNA

摘要 长链非编码RNA（lncRNA）在多种细胞过程中发挥关键作用， 包括转录和转录后调节。Evf 2超保守 lncRNA是一种转录调节lncRNA，从Dlx 5/6超保守的 增强子（UCE）在发育中的前脑。与参与印记的lncRNA不同， Evf 2单方向调控基因表达，对靶基因表达有正、负调控作用 基因. Evf 2与87种蛋白质和对照形成核糖核蛋白复合物（Evf 2-RNP） 影响中间神经元多样性和癫痫发作的基因调控网络的表达 易感性，通过涉及转录因子募集的机制， 以及控制表观遗传修饰和染色体拓扑结构。 对Evf 2亚核定位的研究是了解其功能的关键。 功能Evf 2在每个核中形成一个或两个RNA云。Evf 2-RNP蛋白的一个子集 定位于与Evf 2 RNA云共定位的病灶（蛋白池）中。的共定位 具有Dlx 5/6 UCE和Evf 2调节基因的Evf 2-RNP，揭示了重要的 关于Evf 2依赖性基因调控的机制信息。有趣的是，Evf 2不是 始终保留在其表达位点（Dlx 5/6 UCE），但可与Evf 2相互作用。 调节靶点独立于UCE，这表明Evf 2与 染色体是动态的。 Evf 2-RNP在单细胞水平上的可视化提供了必要的信息 用于理解时空分辨率下的Evf 2调节机制。然而，在这方面， 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.在这个建议中，我们将利用最新的进展，活细胞成像的RNA 用于活细胞中Evf 2的可视化。我们将为这项研究建立小鼠模型， Evf 2云的形成，Evf 2云动力学和Evf 2-RNP形成。本研究将 回答关键问题，以促进对机制的认识， 通过lncRNA进行转录调控。

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.