Mechanisms of gene silencing induced by long noncoding RNAs

长链非编码RNA诱导基因沉默的机制

• 批准号: 9218640
• 负责人: Joseph Mauro Calabrese
• 金额: $ 32.2万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-23 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9218640
• 关键词: Affect; Algorithms; Attenuated; Biological; Biological Assay; Cancer Etiology; Cell physiology; Chemicals; Chromatin; Code; Computing Methodologies; DNA Transposable Elements; Data; Dependency; Development; Disease; Elements; Event; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Hereditary Disease; Human; Human Genome; Knowledge; Link; Live Birth; Messenger RNA; Methods; Molecular; Physiological; Play; Process; Proteins; RNA; Recurrence; Repression; Research; Resolution; Role; Structure; Testing; Trans-Activators; Transcriptional Regulation; Untranslated RNA; Work; X Inactivation; design; experimental study; in vivo; insight; novel

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.

摘要 长链非编码RNA（lncRNA）通过抑制哺乳动物的生长发育， 基因连锁（相邻）蛋白质编码基因的转录。典型的抑制性lncRNA是Xist， 它使几乎所有的基因沿着不活跃的X染色体沉默。其他lncRNA，如Kcnq1ot1和Airn， 在更小的基因组间隔中沉默相邻基因，并可能通过相关机制发挥作用。 总的来说，由抑制性lncRNA的功能改变引起的基因表达失调可以导致 癌症和遗传性疾病影响到大约千分之一的活产婴儿。减弱或增强免疫原性的方法 这种lncRNA的生物学活性可以提供新的手段来控制疾病相关基因的表达， 基因组区域。然而，抑制性lncRNA沉默其靶基因的机制 仍然定义不清，并且尚不清楚如何调节它们的沉默潜力。的 我们研究的长期目标是：（i）确定lncRNA调节的分子机制 基因表达和（ii）应用这些知识来促进人类健康。为此，我们最近 开发了一种还原分析来研究lncRNA诱导的基因沉默的机制。使用该分析，我们 发现了Xist lncRNA的一种新功能，我们假设这是其基因沉默能力所必需的。在 同时，我们开发了一种新的方法来量化lncRNA的序列含量，可以用来预测 lncRNA功能。据我们所知，该算法是同类算法中的第一个，它允许合理设计Xist类 合成lncRNA，并揭示了Xist和数百个其他物种之间共有的重复序列特征， lncRNA，包括许多已知具有抑制活性的lncRNA。最后，通过对Xist的化学分析， 我们发现了这个序列特征的结构背景。这些数据支持我们 中心假设，具有共享序列标记的lncRNA通过 共同机制。为了验证这一假设，我们将：确定Xist的小说的生理后果 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lncRNA沉默所需的反式作用因子（Aim 2），并确定Xist和相关lncRNA沉默所需的反式作用因子。 由Xist样lncRNA诱导的沉默（Aim 3）。我们是唯一适合追求这些目标，因为我们的 专业知识，我们的合作伙伴的专业知识，以及我们的应用程序中描述的数据。在我们的 工作，我们将开发计算和实验方法，可用于预测和研究 任何lncRNA的功能。我们的研究将为lncRNA在细胞生理学中的作用提供新的见解 并可能表明抑制性lncRNA的蛋白质和结构依赖性， 用于控制人类基因组中对正常健康至关重要的区域的转录， 发展