Integrated analysis of cis-acting translocated RNAs (INTACTRNA)

长基因间非编码RNA的一个亚家族，在本文中称为ict（顺式易位）RNA，已经进化出从其合成位点扩散到大染色体结构域或整个染色体内的反义基因的能力。虽然在定义ictRNAs如何沉默基因方面取得了很好的进展，但对它们顺式积累和传播的基础却知之甚少。基于不同的ictRNAs具有共同的起源和作用途径的证据，INTACtRNA将使用一种综合方法来比较和对比Xist和Rsx的活性，这些ictRNAs已经进化到使哺乳动物的X染色体沉默，而Knq 1 ot 1和Airn则使大约15-20 Mb的较小结构域内的基因沉默。INTACTRNA包括三个相互关联的工作包：首先，我们将研究ictRNAs如何逃避核输出并锚定到它们的起源染色体上，应用先进的基因组学，基因组工程和蛋白质降解子策略。第二，我们将探索ictRNAs是如何从它们的合成位点扩散的，并研究调节它们丰度的反馈机制。在这里，我们将优化和应用Nuc-BRITE，这是我们开发的一种新方法，用于活细胞成像和跟踪核RNA。第三，我们将调查什么定义的染色体位点的ictRNAs易位，并建立在以前的工作，染色体拓扑结构在这一过程中的作用。对于这些实验，我们将开发和应用另一种新的方法，sRNA-DamID，用于在高分辨率和单细胞水平上定位关联位点。此外，我们将实施先进的成像方法来连续分析染色体相互作用和ictRNA易位。INTACtRNA将导致我们对控制基因表达过程的理解取得突破性进展，并将进一步促进基因组学和先进细胞成像领域新的最先进方法/技术的开发和应用。

A sub-family of long intergenic non-coding RNAs, referred to herein as ict (in cis translocated) RNAs have evolved the capability to spread from their site of synthesis to inactivate genes within large chromosomal domains or entire chromosomes. Whilst there has been good progress towards defining how ictRNAs silence genes, the basis for their accumulation and spread in cis is poorly understood. Based on evidence that different ictRNAs share common origins and pathways of action, INTACTRNA will use an integrated approach to compare and contrast the activity of Xist and Rsx, ictRNAs that have evolved to silence the X chromosome in mammals, and Knq1ot1 and Airn, ictRNAs that silence genes within smaller domains of around 15-20 Mb. INTACTRNA comprises three interlinked work packages: First, we will investigate how ictRNAs evade nuclear export and become anchored to their chromosome of origin, applying advanced genomics, genome engineering and protein degron strategies. Second, we will probe how ictRNAs spread from their site of synthesis and investigate the feedback mechanisms that regulate their abundance. Here we will optimise and apply Nuc-BRITE, a new method that we have developed, for live-cell imaging and tracking of nuclear RNAs. Thirdly, we will investigate what defines the chromosomal sites to which ictRNAs translocate, and building on prior work, the role of chromosome topology in this process. For these experiments, we will develop and apply another new methodology, sRNA-DamID, for mapping association sites at high resolution and at the single-cell level. In addition we will implement advanced imaging approaches to consecutively analyse both chromosome interactions and ictRNA translocations.INTACTRNA will lead to ground-breaking advances in our understanding of processes controlling gene expression and will further catalyse the development and application of new state-of-the-art methodologies/technologies in genomics and advanced cellular imaging.