lncRNAs as Organizers of and Bridges Between Proteins and DNA

项目摘要 在过去十年中，大规模测序工作出现了一个意想不到的发展- 发现了广泛的全基因组转录。虽然人类基因组计划揭示了 意想不到的一小部分基因组专用于蛋白质编码基因，ENCODE和相关的 项目显示，至少70%的基因组是活跃转录的。这导致了一个新的发现 这类RNA被称为长链非编码RNA或lncRNA。迅速积累的证据有力地表明 这些lncRNA作为RNA具有重要的自主活性。新出现的功能主要是 在发育、分化和多能性等与人类健康密切相关的过程中发挥作用。因此，在本发明中， 建立对lncRNA作用机制的理解是生物学中高度优先的前沿。 为了实现对lncRNA功能的分子理解的长期目标，我们使用了一组 生物化学和结构方法来阐明lncRNA如何组织细胞核的各个方面来调节 并协调染色质表达。 在我们提出的研究计划的第一个目标中，hnRNP U之间相互作用的分子基础 并将研究Xist和Firre RNA。这些lncRNA是X染色体失活所必需的 （Xist）和脂肪形成（Firre），使用需要它们与hnRNP U直接相互作用的机制， 将lncRNA导入正确的染色体或基因座。hnRNP U的RNA结合RGG结构域的选择性结合 将使用“自下而上”和“自上而下”的生物化学方法和结构相结合进行探索 了解蛋白质-RNA识别的分子细节的方法，侧重于关键但很差的 了解RGG结构域。这些研究的结果将阐明许多RNA-蛋白质相互作用 由RGG结构域介导，其是人RNA代谢的中心。 第二个目标是通过研究两个相互作用的lncRNA功能的诱饵/引导模型 糖皮质激素受体（GR）和Sox 2等关键转录因子与lncRNA一起被提出， 调节这些蛋白质的特异性和调节活性。根据这一模式， 启动子和增强子上的转录可以滴定转录因子，使其不与dsDNA结合， 位点，或者相反地，有助于募集转录因子并将其定位于靶。为了揭示序列和/或 这些双链DNA结合蛋白可以识别的lncRNA的结构，一种基于体外选择的方法 将被使用，并且结果与转录组学研究相关。同时，传统的生物化学和 结构方法将用于了解这些蛋白质-RNA相互作用的分子细节， 已知的lncRNA靶点这些研究将产生直接的见解，如何关键转录因子， 传统上被认为是唯一的双链DNA结合因子也与RNA相互作用，作为其基因的关键部分 监管机制

Project Summary One of the unexpected developments in the last decade emerged from large scale sequencing efforts - the discovery of widespread genome-wide transcription. While the human genome project revealed an unexpectedly small fraction of the genome dedicated to protein-coding genes, the ENCODE and related projects revealed that at least 70% of the genome is actively transcribed. This led to the discovery of a new class of RNAs known as long non-coding RNAs or lncRNAs. Rapidly accumulating evidence strongly suggests that these lncRNAs have important autonomous activities as RNAs. The emerging functions are predominantly in development, differentiation and pluripotency, processes with critical links to human health. Thus, establishing an understanding of the mechanism of action of lncRNAs is a high priority frontier in biology. Towards the long term-goal of producing a molecular understanding of lncRNA function, we are using a set of biochemical and structural approaches to elucidate how lncRNAs organize aspects of the nucleus to regulate and coordinate chromatin expression. In the first Aim of our proposed research program, the molecular basis of the interaction between hnRNP U and the Xist and Firre RNAs will be investigated. These lncRNAs are essential for X-chromosome inactivation (Xist) and adipogenesis (Firre), using mechanisms requiring their direct interaction with hnRNP U that localizes the lncRNA to the correct chromosome or loci. Selective binding of the RNA-binding RGG domain of hnRNP U will be explored using a combination of "bottom-up" and "top-down" biochemical approaches and structural approaches to understand the molecular details of protein-RNA recognition, focusing on the critical but poorly understood RGG domain. The results of these studies will illuminate a number of RNA-protein interactions mediated by RGG domains that are central to human RNA metabolism. The second Aim focuses on the decoy/guide model for lncRNA function by investigating the interactions of two key transcription factors, glucocorticoid receptor (GR) and Sox2, with the lncRNAs that are proposed to modulate the specificity and regulatory activity of these proteins. According to this model, pervasive transcription at promoters and enhancers can either titrate away a transcription factor from its dsDNA-binding site or, conversely, help to recruit and localize a transcription factor to a target. To reveal the sequences and/or structures of lncRNAs that these dsDNA-binding proteins can recognize, an in vitro selection based approach will be used and the results correlated with transcriptomic studies. In parallel, traditional biochemical and structural approaches will be used to understand the molecular details of these protein-RNA interactions with known lncRNA targets. These studies will yield direct insights into how key transcription factors that have classically been regarded as solely dsDNA-binding factors also interact with RNA as a critical part of their gene regulatory mechanism.