Mouse models for live-cell imaging of endogenous Evf2 lncRNA

点击翻译按钮获取中文摘要

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.