The Structural Basis of PRC2 Recruitment by lncRNA

lncRNA招募PRC2的结构基础

• 批准号: 8977246
• 负责人: Thayne Henderson Dickey
• 金额: $ 5.24万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977246
• 关键词: Acylation; Affect; Affinity; Alzheimer' s Disease; Binding; Biochemical; Biological Assay; Biological Models; Boxing; Calorimetry; Cells; Chemicals; Chromatin; Code; Complex; Data; Deposition; Development; Disease; Dosage Compensation (Genetics); Drosophila genus; Electrophoretic Mobility Shift Assay; Elements; Female; Gene Expression; Genetic; Goals; Histone Acetylation; Human Genome; Hydroxyl Radical; In Vitro; Literature; Location; Malignant Neoplasms; Mammals; Methods; Modeling; Molecular; Molecular Sieve Chromatography; Neoplasm Metastasis; Neurodegenerative Disorders; Primer Extension; Production; Protein Footprinting; Proteins; RNA; RNA Folding; Recruitment Activity; Reporting; Resolution; Sampling; Solutions; Specificity; Structure; System; Techniques; Testing; Therapeutic; Tissues; Titrations; Transcript; Untranslated RNA; X Chromosome; base; design; dimethyl sulfate; fly; histone methyltransferase; histone modification; improved; infancy; insight; novel therapeutics; protein structure; public health relevance; research study; theories

 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.

 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等疾病有关。尽管lncRNA很重要，但我们只了解其中少数的功能。即使对于这些经过研究的少数人，我们的理解也停留在遗传或分子水平上，并且我们缺乏对它们如何发挥作用的详细结构理解。该结构信息对于验证提议的至关重要 机制，理解和预测未经研究的 lncRNA 的功能，并可能以 lncRNA 为治疗目的。 lncRNA 最常见的功能之一是招募染色质修饰复合物。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（例如癌症转移）中的意义一致。也许最具特征的lncRNA，Xist及其部分重叠的5'转录本repA，通过在雌性哺乳动物的剂量补偿期间招募PRC2来沉默X染色体来以这种方式发挥作用。其他 lncRNA，例如 HOTAIR，也通过与 5' 结构域相互作用来招募 PRC2，这表明了一种全局作用机制。然而，最近的文献报道对于 PRC2 募集的机制和特异性存在不同意见。因此，我们将另外利用果蝇中的类似模型系统来深入了解 lncRNA 募集组蛋白修饰复合物的结构基础。 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/PRC2 系统的方式将 MSL 复合物招募到 X 染色体上。然而，在果蝇中，该复合物的成分已得到很好的表征，并且已提出两种蛋白质成分与特定的 RNA 元件特异性相互作用。这些模型将使用体外生化方法（例如电泳迁移率变动测定和等温滴定量热法）进行验证。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。 第二个目标是对 lncRNA repA 进行结构表征。 repA将在体外转录， 但随后将通过尺寸排阻色谱法进行纯化，无需传统的变性步骤。这种生产方法已被证明可以生产均质、折叠良好的 RNA，并可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那些与 PRC2 特异性相互作用的 RNA 结构元件。这些目标将共同阐明 lncRNA 和组蛋白修饰复合物之间相互作用的保守机制。