Mechanisms of gene silencing induced by long noncoding RNAs

摘要 长非编码RNA(LncRNAs)通过抑制基因转录，在人类健康中发挥重要作用。 相互关联的基因。LncRNAs沉默靶基因表达的改变会导致毁灭性的遗传 疾病，如Angelman、Beckwith-Wiedemann和Rett综合征，以及癌症和免疫 综合症。最有效和研究最多的抑制性lncRNA，Xist，在整个 X染色体在女性体内实现X-失活。尽管如此，Xist和其他lncRNA的机制 沉默他们的目标基因还不够了解。事实上，在RNA序列的水平上，还不清楚是什么 区分抑制性和缺乏抑制性的lncRNA，严重限制了我们对 机制和我们在其他lncRNA中识别抑制功能的能力。这项研究的长期目标是 是为了确定RNA序列如何指定lncRNAs中的抑制功能，并确定其机制 通过它来实施镇压。我们过去五年的研究使我们更接近这些目标。 最值得注意的是，我们开发了一种非线性序列比较的方法，它证明了尽管 由于缺乏线性同源性，抑制性lncRNA富含相似的序列基序，这表明 他们如何对镇压进行编码的模型。我们发现抑制性的lncRNA表现出丰富的关联性 具有特定的蛋白质，支持一般模型。我们开发了新的方法来绘制地图、量化和 操纵lncRNA与蛋白质的相互作用。最后，我们的数据提供了一个解释，说明了 Xist的转录和Xist诱导基因的早期阶段都需要Xist的5美分末端 沉默。根据我们的发现，我们假设具有共同序列属性的lncRNAs抑制 通过相关机制转录。在下一个资助期，我们将用三个例子来检验这一假设 具体目标：(1)确定单个结构域如何协调XIST产生和靶基因沉默； (2)确定RNA-蛋白质相互作用和RNA结构如何在三个进化阶段中指定抑制功能 无关的lncRNA；以及(3)开发并严格验证扫描转录本以检测lncRNAs的新方法 和具有类似功能的lncRNA结构域。这些进展将加速RNA的发现-- 调解对发育和疾病至关重要的事件。

ABSTRACT Long noncoding RNAs (lncRNAs) play essential roles in human health by repressing transcription of genetically- linked genes. Altered expression of the genes that lncRNAs target for silencing drives devastating genetic disorders such as Angelman, Beckwith-Wiedemann, and Rett Syndromes, as well as cancers and immune syndromes. The most potent and most studied repressive lncRNA, Xist, silences transcription across one entire X chromosome to achieve X-inactivation in females. Still, the mechanisms by which Xist and other lncRNAs silence their target genes are insufficiently understood. Indeed, at the level of RNA sequence, it is unclear what distinguishes repressive lncRNAs from those that lack repressive activity, severely limiting our understanding of mechanism and our ability to identify repressive function in other lncRNAs. The long-term goals of this research are to determine how RNA sequence specifies repressive function in lncRNAs and determine the mechanisms by which the repression is carried out. Our studies over the past five years have brought us closer to these goals. Most notably, we developed a method of non-linear sequence comparison which demonstrated that despite lacking linear homology, repressive lncRNAs are enriched in similar sequence motifs, suggesting a general model for how they encode repression. We discovered that repressive lncRNAs exhibit enriched associations with specific proteins, supporting the general model. We developed new approaches to map, quantify, and manipulate lncRNA-protein interactions. Lastly, our data provide an explanation for how a single domain in the 5¢ end of Xist is required both for the transcription of Xist and for the earliest stages of Xist-induced gene silencing. On the basis of our findings, we hypothesize that lncRNAs with shared sequence properties repress transcription by related mechanisms. During the next funding period, we will test this hypothesis with three Specific Aims: (1) determine how a single domain orchestrates both Xist production and target-gene silencing; (2) determine how RNA-protein interactions and RNA structure specify repressive function in three evolutionarily unrelated lncRNAs; and (3) develop and rigorously validate a new approach to scan transcriptomes for lncRNAs and lncRNA domains that harbor analogous functions. These advances will accelerate the discovery of RNA- mediated events that are essential for development and disease.