The Structural Basis of PRC2 Recruitment by lncRNA

 描述（由申请人提供）：人类基因组编码数千种长非编码RNA（lncRNA），其中许多与从癌症到神经退行性疾病的疾病有关。尽管lncRNA的重要性，我们只了解其中少数的功能。即使对这些已经研究过的少数几个，我们的理解也停留在遗传或分子水平上，我们缺乏对它们如何发挥作用的详细结构理解。这种结构信息对于验证建议的 机制，理解和预测未研究的lncRNA的功能，并潜在地靶向lncRNA用于治疗目的。 lncRNA最常见的功能之一是募集染色质修饰复合物。这种功能与它们的组织特异性表达及其在与细胞分化相关的疾病如癌症转移中的意义一致。也许最佳表征的lncRNA Xist及其部分重叠的5'转录物repA以这种方式通过募集PRC 2来在雌性哺乳动物中的剂量补偿期间沉默X染色体而起作用。另外的lncRNA，如HOTAIR，也通过与5'结构域的相互作用募集PRC 2，这表明了一种全局作用机制。然而，最近的文献报道不同意PRC 2招募的机制和特异性。因此，我们将另外利用苍蝇中的类似模型系统来深入了解组蛋白修饰复合物的lncRNA募集的结构基础。 第一个目标是确定果蝇中roX lncRNA和MSL组蛋白修饰复合物之间特异性相互作用的结构基础。roX lncRNA以类似于Xist/PRC 2系统的方式将MSL复合物募集到X染色体。然而，在果蝇中，这种复合物的组分已经被很好地表征，并且已经提出了两种蛋白质组分与定义的RNA元件特异性相互作用。这些模型将使用体外生物化学方法进行验证，如电泳迁移率变动分析和等温滴定量热法。最终，这些生化数据将有助于解决负责赋予roX/MSL复合物特异性的复合物的X射线晶体结构。 第二个目标是在结构上表征lncRNA repA。repA将在体外转录， 但随后将通过尺寸排阻色谱法纯化，而无需传统的变性步骤。这种生产方法已被证明可以产生同质的，折叠良好的RNA，适合通过化学探测进行结构表征。蛋白质足迹分析也将用于确定与PRC 2特异性相互作用的那些RNA结构元件。总之，这些目标将阐明lncRNA和组蛋白修饰复合物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.