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Functional dissection of the Tug1 long noncoding RNA locus

Tug1 长非编码 RNA 基因座的功能剖析

基本信息

批准号：
424761055
负责人：
Dr. Christian Much
金额：
--
依托单位：
BioFrontiers Institute
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2019
资助国家：
德国
起止时间：
2018-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/424761055?language=en
关键词：
Functional dissection Tug1 long noncoding

项目摘要

Several long noncoding RNAs (lncRNAs) have been ascribed a function in the regulation of gene expression. But even though thousands of lncRNAs exist, many of them are merely transcriptional byproducts that lack an RNA-dependent role. Therefore, lncRNA genes of regulatory importance must preferentially act in different ways to exert their functions. We set out to study the lncRNA locus Tug1, which in contrast to many other lncRNAs is ubiquitously expressed and highly conserved between mouse and human. Remarkably, we found that the Tug1 locus plays an indispensable role in male fertility, as Tug1-deleted mice are completely sterile due to spermatogenesis defects. Our genetic and molecular analyses indicate that the Tug1 locus functions through multiple mechanisms, involving DNA-, RNA- and protein-mediated effects. We gathered experimental evidence that the Tug1 locus harbors a repressive DNA element acting on neighboring genes, that it produces an RNA that regulates the expression of distant genes, and that it might encode a peptide of unknown function. However, how these components contribute to the function and physiological significance of the Tug1 locus remains unknown.Here I propose to functionally dissect the Tug1 locus with the aim to understand its mechanisms of action. For this purpose, I will analyze the contributions of DNA, RNA and protein to the regulatory logic of the Tug1 locus using mouse embryonic stem cells as a model system. The repressive DNA element will be characterized by testing multiple sequences derived from the Tug1 locus for their gene silencing activity and their ability to recruit repressive protein complexes. In order to define how the Tug1 RNA regulates its distant gene targets, I will use loss- and gain-of-function experiments to define the true targets of Tug1 as well as identify specific RNA domains and RNA-binding proteins that are important for gene regulation. The protein-coding potential of the Tug1 gene will be tested through bioinformatic and experimental approaches, and identified TUG1 peptides will be investigated for their ability to regulate gene expression and other processes in the cell.Collectively, these experiments will define the multifaceted features of the Tug1 locus and elucidate whether they converge on a single function or act independently in different biological pathways. The complex characteristics of the Tug1 locus might be more common than previously thought and help explain the functionality of many other lncRNA genes.

一些长链非编码RNA（lncRNA）被认为在基因表达调控中起作用。但是，即使存在成千上万的lncRNA，其中许多只是转录副产物，缺乏RNA依赖的作用。因此，具有调控重要性的lncRNA基因必须优先以不同的方式发挥其功能。我们着手研究lncRNA位点Tug1，与许多其他lncRNA相反，它在小鼠和人类之间普遍表达且高度保守。值得注意的是，我们发现Tug1基因座在雄性生育能力中起着不可或缺的作用，因为Tug1缺失的小鼠由于精子发生缺陷而完全不育。我们的遗传和分子分析表明，Tug1基因座通过多种机制发挥作用，涉及DNA，RNA和蛋白质介导的作用。我们收集的实验证据表明，Tug 1位点含有一个抑制性DNA元件，作用于邻近基因，它产生一种RNA，调节远端基因的表达，它可能编码一种功能未知的肽。然而，这些组件如何有助于Tug1基因座的功能和生理意义仍然未知。在这里，我建议功能解剖的Tug1基因座的目的是了解其作用机制。为此，我将使用小鼠胚胎干细胞作为模型系统，分析DNA，RNA和蛋白质对Tug1基因座调控逻辑的贡献。将通过测试源自Tug1基因座的多个序列的基因沉默活性及其募集阻遏蛋白复合物的能力来表征阻遏DNA元件。为了确定Tug1 RNA如何调节其远端基因靶点，我将使用功能丧失和获得实验来确定Tug1的真正靶点，并确定对基因调控重要的特定RNA结构域和RNA结合蛋白。Tug1基因的蛋白质编码潜力将通过生物信息学和实验方法进行测试，并确定Tug1肽将研究其调节基因表达和细胞中其他过程的能力。总的来说，这些实验将定义Tug1基因座的多方面特征，并阐明它们是否收敛于单一功能或在不同的生物学途径中独立发挥作用。Tug1基因座的复杂特征可能比以前认为的更常见，并有助于解释许多其他lncRNA基因的功能。