Mechanisms of gene silencing induced by long noncoding RNAs

摘要 长链非编码RNA（lncRNA）通过抑制基因转录在人类健康中发挥重要作用。 连锁基因lncRNA靶向沉默的基因表达的改变驱动了破坏性的遗传学改变， 疾病，如Angelman，Beckwith-Wiedemann和Rett Syndrome，以及癌症和免疫性疾病。 综合征最有效和研究最多的抑制性lncRNA，Xist，沉默转录在一个完整的 X染色体，以实现女性的X失活。尽管如此，Xist和其他lncRNAs 沉默它们的靶基因是不充分理解的。事实上，在RNA序列的水平上，还不清楚 将抑制性lncRNA与那些缺乏抑制活性的lncRNA区分开来，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lncRNA的抑制功能，并确定其机制， 镇压的方式。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，该方法表明，尽管 由于缺乏线性同源性，抑制性lncRNA富含相似的序列基序，这表明一般的 它们如何编码抑制的模型。我们发现抑制性lncRNAs表现出丰富的关联 与特定的蛋白质，支持一般模型。我们开发了新的方法来映射，量化， 操纵lncRNA-蛋白质相互作用。最后，我们的数据提供了一个解释，为什么一个单一的领域， Xist的5 ′端是Xist转录和Xist诱导基因表达的最早阶段所必需的 沉默基于我们的发现，我们假设具有共同序列特性的lncRNA抑制了 转录相关机制。在下一个融资期间，我们将用三个 具体目的：（1）确定一个结构域如何协调Xist的产生和靶基因的沉默; (2)确定RNA-蛋白质相互作用和RNA结构如何在三个进化上指定抑制功能 不相关的lncRNA;（3）开发并严格验证一种扫描lncRNA转录组的新方法 和具有类似功能的lncRNA结构域。这些进展将加速RNA的发现- 介导的事件对发育和疾病至关重要。

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.