Mechanisms of gene silencing induced by long noncoding RNAs

摘要 长非编码RNA(LncRNAs)在哺乳动物发育过程中发挥着重要的作用。 基因连锁的(相邻的)蛋白质编码基因的转录。原型抑制性lncRNA是Xist， 这会使非活跃X染色体上的几乎所有基因沉默。其他lncRNA，如Kcnq1ot1和Airn， 在较小的基因组间隔内沉默相邻基因，并可能通过相关机制发挥作用。 总的来说，由抑制性lncRNAs功能改变引起的基因表达失调可导致 癌症和遗传疾病，大约每1000名活产中就有一名受到影响。减弱或增强的方法 这些lncRNAs的生物学活性可以为控制疾病相关基因的表达提供新的手段。 基因组的区域。然而，抑制性lncRNA使其靶基因沉默的机制 仍然没有明确的定义，也不清楚如何调节它们的沉默潜力。这个 我们研究的长期目标是(I)确定lncRNAs调控的分子机制 基因表达和(Ii)将这一知识应用于促进人类健康。为此，我们最近 开发了一种还原论方法来研究lncRNA诱导的基因沉默的机制。使用化验，我们 发现了Xist lncRNA的一种新功能，我们推测这是其基因沉默能力所必需的。在……里面 同时，我们开发了一种新的方法来量化lncRNAs的序列含量，可以用来预测 LncRNA功能。这个算法--据我们所知是第一个--允许类Xist的合理设计 合成的lncRNA，并发现了Xist和数百个其他 InncRNA，包括许多已知具有抑制活性的RNA。最后，通过对Xist‘s的化学探测 二级结构我们发现了这个序列签名的结构背景。这些数据支持我们的 中心假设，具有共同序列签名的lncRNAs通过 共同的机制。为了验证这一假设，我们将：确定希斯特小说的生理后果 功能及其发生的机制(目标1)，确定序列和结构特征 Xist和相关的lncRNAs沉默所需的(目标2)，并确定所需的反式作用因子 Xist-like lncRNAs诱导的沉默(目标3)。我们非常适合追求这些目标，因为我们的 专业知识、我们的合作者的专业知识以及我们的应用程序中描述的数据。在我们的过程中 工作中，我们将开发可用于预测和研究的计算和实验方法 任何lncRNA的功能。我们的研究将对lncRNAs在细胞生理学中的作用提供新的见解。 和疾病，并可能提示抑制lncRNA的蛋白质和结构依赖性，这可能是 被用来控制人类基因组中对正常健康和 发展。

ABSTRACT Long noncoding RNAs (lncRNAs) play essential roles in mammalian development by repressing transcription of genetically linked (adjacent) protein coding genes. The archetypal repressive lncRNA is Xist, which silences nearly all genes along the inactive X chromosome. Other lncRNAs, such as Kcnq1ot1 and Airn, silence adjacent genes in smaller genomic intervals, and may function through related mechanisms. Collectively, dysregulated gene expression caused by altered function of repressive lncRNAs can cause cancers and genetic disorders that affect roughly 1 in 1,000 live births. Methods to attenuate or potentiate the biological activity of such lncRNAs could provide new means to control gene expression in disease relevant regions of the genome. However, the mechanisms by which repressive lncRNAs silence their target genes remain poorly defined, and it is unclear how modulation of their silencing potential might be achieved. The long-term goals of our research are (i) to determine the molecular mechanisms by which lncRNAs regulate gene expression and (ii) to apply this knowledge to advance human health. To these ends, we recently developed a reductionist assay to study mechanisms of lncRNA-induced gene silencing. Using the assay, we discovered a novel function of the Xist lncRNA that we hypothesize is essential for its gene silencing ability. In parallel, we developed a novel method to quantify sequence content of lncRNAs that can be used to predict lncRNA function. This algorithm – to our knowledge the first of its kind – allowed rational design of Xist-like synthetic lncRNAs and uncovered a recurrent sequence signature shared between Xist and hundreds of other lncRNAs, including many known to have repressive activity. Lastly, through chemical probing of Xist's secondary structure we discovered a structural context for this sequence signature. These data support our central hypothesis, that lncRNAs with a shared sequence signature silence gene expression through a common mechanism. To test this hypothesis, we will: determine the physiological consequences of Xist's novel function and the mechanism by which it occurs (Aim 1), determine the sequence and structural signatures required for silencing by Xist and related lncRNAs (Aim 2), and determine the trans-acting factors required for silencing induced by Xist-like lncRNAs (Aim 3). We are uniquely suited to pursue these objectives given our expertise, the expertise of our collaborators, and the data described in our application. In the course of our work, we will develop computational and experimental methods that can be applied to predict and study function of any lncRNA. Our study will provide novel insights into the roles of lncRNAs in cellular physiology and disease and may suggest protein and structural dependencies of repressive lncRNAs that can be exploited to control transcription in regions of the human genome that are essential for normal health and development.