The Structural Basis of PRC2 Recruitment by lncRNA

 描述(申请人提供)：人类基因组编码数以千计的长非编码RNA(LncRNAs)，其中许多与从癌症到神经退行性疾病等疾病有关。尽管lncRNA很重要，但我们只了解其中一小部分的功能。即使对于这些已被研究过的精选少数几个，我们的理解存在于基因或分子水平，我们缺乏对它们如何发挥作用的详细结构理解。此结构信息对于验证建议的 机制，了解和预测未研究的lncRNAs的功能，以及潜在的靶向治疗目的的lncRNAs。LncRNAs最常见的功能之一是募集染色质修饰复合体。这一功能与它们在组织中的特异性表达以及它们在与细胞分化相关的疾病中的意义是一致的，例如癌症转移。也许最具特性的lncRNAs，Xist及其部分重叠的5‘转录REPA，通过招募PRC2在雌性哺乳动物的剂量补偿期间沉默X-染色体，以这种方式发挥作用。其他的lncRNAs，如HOTAIR，也通过与5‘结构域的相互作用招募PRC2，这表明了一种全球的作用机制。然而，最近的文献报道不同意PRC2招募的机制和特异性。因此，我们将另外利用一个类似的果蝇模型系统来深入了解组蛋白修饰复合体的lncRNA募集的结构基础。第一个目标是确定果蝇中Rox lncRNAs和MSL组蛋白修饰复合体之间特定相互作用的结构基础。Rox lncRNAs以类似于Xist/PRC2系统的方式将MSL复合体招募到X染色体上。然而，在果蝇中，这种复合体的成分已经被很好地表征，并且有两个蛋白质成分被提出与一个定义的RNA元件特异地相互作用。这些模型将使用体外生化方法进行验证，如电泳迁移率变化分析和等温滴定量热法。最终，这些生化数据将有助于解决导致ROX/MSL复合体中特异性的复合体的X射线晶体结构。第二个目标是对lncRNA REPA的结构特征进行表征。REPA将在体外转录， 但随后将通过尺寸排除层析进行纯化，而不需要传统的变性步骤。这种生产方法已被证明能生产出均一、折叠良好的RNA，并能通过化学探针进行结构表征。蛋白质足迹分析也将被用来确定那些与PRC2特异相互作用的RNA结构元件。总之，这些目标将阐明lncRNAs和组蛋白修饰复合体之间相互作用的保守机制。

 DESCRIPTION (provided by applicant): The human genome codes for thousands of long noncoding RNAs (lncRNAs), many of which are implicated in diseases ranging from cancer to neurodegenerative disorders. Despite the importance of lncRNAs, we only understand the function of a handful of them. Even for these select few that have been studied, our understanding lies at the genetic or molecular level and we lack a detailed structural understanding of how they function. This structural information is critical for validating proposed mechanisms, understanding and predicting the function of unstudied lncRNAs, and potentially targeting lncRNAs for therapeutic purposes. One of the most common functions of lncRNAs is the recruitment of chromatin modifying complexes. This function is consistent with their tissue specific expression and their implication in diseases related to cellular differentiation, such as cancer metastasis. Perhaps the best-characterized lncRNAs, Xist and its partially overlapping 5' transcript repA, function in this manner by recruiting PRC2 to silence the X- chromosome during dosage compensation in female mammals. Additional lncRNAs, such as HOTAIR, also recruit PRC2 via interaction with a 5' domain, suggesting a global mechanism of action. Recent reports in the literature, however, disagree about the mechanism and specificity of PRC2 recruitment. Thus, we will additionally utilize an analogous model system in flies to gain insight into the structural basis of lncRNA recruitment of histone modification complexes. The first aim will be to determine the structural basis for the specific interaction between the roX lncRNAs and the MSL histone modification complex in Drosophila. The roX lncRNAs recruit the MSL complex to the X-chromosome in a fashion akin to the Xist/PRC2 system. In flies, however, the components of this complex have been well characterized and two protein components have been proposed to specifically interact with a defined RNA element. These models will be validated using in vitro biochemical approaches such as electrophoretic mobility shift assays and isothermal titration calorimetry. Ultimately, these biochemical data will facilitate the solution of the x-ray crystal structure of the complex responsible for conferring specificity in the roX/MSL complex. The second aim will be to structurally characterize the lncRNA repA. repA will be transcribed in vitro, but will be subsequently purified by size-exclusion chromatography without the traditional denaturation step. This production method has been shown to produce homogenous, well-folded RNA amenable to structural characterization by chemical probing. Protein footprinting assays will also be used to determine those RNA structural elements that specifically interact with PRC2. Together, these aims will elucidate the conserved mechanisms of interaction between lncRNAs and histone modification complexes.