lncRNAs as Organizers of and Bridges Between Proteins and DNA

项目摘要 在过去十年中，大规模测序工作中出现了意想不到的发展-- 发现广泛的全基因组转录。虽然人类基因组计划揭示了 基因组中出人意料的一小部分专门用于蛋白质编码基因，Encode和相关 项目显示，至少70%的基因组是活跃转录的。这导致了一种新的发现 称为长非编码RNA或LncRNAs的一类RNAs。迅速积累的证据有力地表明 这些lncRNAs作为RNAs具有重要的自主活动。新出现的功能主要是 在发展、分化和多能性方面，与人类健康有着关键联系的过程。因此， 建立对lncRNAs作用机制的理解是生物学中的一个高度优先的前沿。 为了实现对lncRNA功能的分子理解的长期目标，我们正在使用一套 用生化和结构方法阐明lncRNAs如何组织细胞核的各个方面进行调控 并协调染色质的表达。 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 并将对Xist和Firre RNA进行调查。这些lncRNAs是X染色体失活所必需的 (Xist)和脂肪生成(Firre)，使用它们需要与定位的hnRNP U直接相互作用的机制 正确的染色体或基因座上的lncRNA。HnRNP U与RNA结合的RGG结构域的选择性结合 将结合使用“自下而上”和“自上而下”的生化方法和结构 理解蛋白质-RNA识别的分子细节的方法，侧重于关键但不好的 了解RGG领域。这些研究的结果将阐明一些RNA-蛋白质的相互作用 由对人类RNA新陈代谢起中心作用的RGG结构域介导。 第二个目的是研究lncRNA功能的诱饵/引导模型，通过研究两者的相互作用 关键的转录因子，糖皮质激素受体(GR)和Sox2，以及被认为是 调节这些蛋白质的特异性和调节活性。根据这个模型，无处不在 启动子和增强子的转录可以滴定出转录因子与dsdna的结合。 或相反，帮助招募转录因子并将其定位于靶标。以揭示序列和/或 基于体外选择的dsDNA结合蛋白可识别的lncRNAs结构 将被使用，结果与转录研究相关。同时，传统的生化和 结构方法将用来理解这些蛋白质-RNA相互作用的分子细节 已知的lncRNA靶标。这些研究将直接深入了解关键转录因子是如何 传统上被认为是唯一的dsDNA结合因子也作为其基因的关键部分与rna相互作用。 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.