Deciphering the mechanism of large ncRNA mediated regulation of cell state

描述（由申请人提供）：我们先前描述了使用活性转录的染色质标记鉴定一类约3500个保守的大基因间非编码RNA（lincRNA）。这些lincRNA在细胞中具有全局功能，并在细胞生长中发挥关键作用。 包括调节多能细胞状态在内的多种生物过程。虽然 虽然lincRNA在功能上是重要的，但它们发挥调节作用的机制目前尚不清楚。由于许多lincRNA同时与多种不同的蛋白质复合物相互作用，一种假设是lincRNA充当“灵活的模块化支架”，将蛋白质复合物聚集在一起形成更大的功能单元。在这个模型中，RNA包含与特定蛋白质复合物相互作用的离散结构域。这些RNA通过结构域的组合，使各个调控组分接近，从而形成独特的调控RNA。在这里，我们建议通过理解lincRNA-蛋白质复合物如何形成、定位于调控靶点并产生表型状态来破译lincRNA介导的调控机制。我们将通过使用基因组方法结合生物化学方法来表征lincRNA-蛋白质相互作用，分子生物学将lincRNA映射到其直接靶点，遗传学方法删除lincRNA结构域并在体内组装修饰的RNA基因，以及计算生物学将这些组件整合到lincRNA介导的基因组调控模型中来解决这些问题。我们将鉴定与lincRNA相互作用的所有蛋白质复合物，确定这些蛋白质相互作用在RNA上发生的位置，并确定这些lincRNA-蛋白质相互作用如何组装（目标1）。我们将确定lincRNA的直接调控靶点，并确定lincRNA如何实现调控特异性（目的2）。我们将确定单个lincRNA-蛋白质相互作用结构域如何发挥作用，以及lincRNA如何通过拼凑功能结构域进行功能组装（目的3）。总之，这些结果将使我们能够理解lincRNA如何利用离散结构域来靶向和调节特定基因组的全部复杂性，并可能允许创建可以执行工程调控作用的合成工程RNA。

DESCRIPTION (provided by applicant): We previously described the identification of a class of ~3500 conserved large intergenic noncoding RNAs (lincRNAs) using a chromatin signature of active transcription. These lincRNAs are globally functional in the cell and play critical roles in diverse biological processes including in the regulation of the pluripotent cell state. While it is now clear that lincRNAs are functionally important, the mechanism by which they carry out their regulatory role is currently unknown. As many of the lincRNAs interact simultaneously with multiple different protein complexes, one hypothesis is that lincRNAs act as 'flexible modular scaffolds' to bring together protein complexes into larger functional units. In this model, RNA contains discrete domains that interact with specific protein complexes. These RNAs, through a combination of domains, bring individual regulatory components into proximity resulting in the formation of a unique regulatory RNA. Here we propose to decipher the mechanism of lincRNA mediated regulation by understanding how lincRNA-Protein complexes form, localize to regulatory targets, and give rise to phenotypic states. We will address these questions by using genomic methods in conjunction with biochemical methods to characterize lincRNA-Protein interactions, molecular biology to map lincRNAs to their direct targets, genetic methods to delete lincRNA domains and assemble modified RNA genes in vivo, and computational biology to integrate these components into a model of lincRNA mediated genome regulation. We will identify all protein-complexes with which lincRNAs interact, determining where these protein interactions occur on RNA, and determining how these lincRNA-Protein interactions assemble (Aim 1). We will identify the direct regulatory targets of lincRNAs and determine how lincRNAs achieve regulatory specificity (Aim 2). We will determine how individual lincRNA-Protein interaction domains function, and how lincRNAs are functionally assembled by piecing together functional domains (Aim 3). Together, these results will allow us to understand the full complexity of how lincRNAs can utilize discrete domains to target and regulate specific sets of genes and may allow the creation of synthetically engineered RNAs that can carry out engineered regulatory roles.