Functional dissection of the Tug1 long noncoding RNA locus

一些长链非编码rna （lncRNAs）被认为在基因表达调控中起作用。但是，尽管存在成千上万的lncrna，其中许多只是转录副产物，缺乏依赖rna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。我们开始研究lncRNA基因座Tug1，与许多其他lncRNA不同，它在小鼠和人之间普遍表达且高度保守。值得注意的是，我们发现Tug1位点在雄性生殖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位点通过多种机制发挥作用，包括DNA、RNA和蛋白质介导的作用。我们收集的实验证据表明，Tug1位点含有一个作用于邻近基因的抑制DNA元件，它产生一种调节远端基因表达的RNA，它可能编码一种功能未知的肽。然而，这些成分如何影响Tug1位点的功能和生理意义仍然未知。在这里，我建议对Tug1基因座进行功能解剖，目的是了解其作用机制。为此，我将以小鼠胚胎干细胞为模型系统，分析DNA、RNA和蛋白质对Tug1位点调控逻辑的贡献。抑制DNA元件将通过测试来自Tug1位点的多个序列来确定其基因沉默活性和招募抑制蛋白复合物的能力。为了确定Tug1 RNA如何调节其远端基因靶标，我将使用功能丧失和功能获得实验来确定Tug1的真正靶标，并确定对基因调控重要的特定RNA结构域和RNA结合蛋白。将通过生物信息学和实验方法测试Tug1基因的蛋白质编码潜力，并研究已鉴定的Tug1肽在细胞中调节基因表达和其他过程的能力。总的来说，这些实验将定义Tug1基因座的多方面特征，并阐明它们是集中在一个单一的功能上，还是在不同的生物学途径中独立作用。Tug1位点的复杂特征可能比以前认为的更普遍，并有助于解释许多其他lncRNA基因的功能。

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.