Large-scale characterization of the function of RNA regulatory elements

项目摘要 一旦RNA在细胞核中转录，它就被RNA结合蛋白结合， 其他调控RNA控制其序列（通过改变剪接），半衰期（通过调节 RNA稳定性和衰变）和翻译（通过介导核糖体起始和 延伸）以及其他RNA加工步骤。单个RBP结合位点可以具有 显著的生理重要性，正如最近的药物例子所强调的那样， Spinraza是一种反义寡核苷酸，可阻断单个RBP：RNA相互作用， 治疗脊髓性肌萎缩症基因组学技术的最新进展 极大地提高了我们识别这些调控RBP相互作用位点的能力， 我们现在有超过100万个这样的相互作用位点的目录，它们是候选RNA 监管要素。然而，很明显，这些元素中只有一小部分真正 作为调节模块，当RBP被调节时，很少有RNA加工的差异。 被推倒或改变因此，筛选这些元素的大规模测定 识别功能的子集是转换这些功能的重要缺失部分， 元素数据库成为一个有用的工具，研究人员有兴趣了解人类是否 基因变异将改变RNA生物学。 在这份提案中，我描述了我的研究小组为解决这一重大问题所做的努力。 两个领域的知识差距： 1.使用正交方法（快速降解RBPs，然后进行基因组学研究） 分析以鉴定RBP和大规模平行报告基因的直接调控靶点 测定）以表征哪些RBP结合位点赋予调节。 2.大规模鉴定snoRNA、snRNA和其他调控靶点 通过改进的基因组学技术来调节RNA。 我在开发实验和计算基因组学方法方面拥有广泛的专业知识， 绘制和理解RNA加工调控网络使我新成立的实验室成为一个 开展这些工作的理想地点，并建立一个改进的全球RNA图片 处理监管景观。此外，它将支持我努力发展一个独立的 研究小组，将奠定基础，以更广泛的努力，以了解人类如何 遗传变异通过RNA加工的错误调节影响疾病。

PROJECT SUMMARY Once an RNA is transcribed in the nucleus, it is bound by RNA binding proteins and other regulatory RNAs which control its sequence (by altering splicing), half-life (by modulating RNA stability and decay) and translation (through mediation of ribosome initiation and elongation) among other RNA processing steps. Individual RBP binding sites can have significant physiological importance, as emphasized by the recent example of the drug Spinraza, which is an antisense oligonucleotide that blocks a single RBP:RNA interaction in order to cure Spinal Muscular Atrophy. Recent advances in genomics techniques have dramatically increased our ability to identify the interaction sites for these regulatory RBPs and RNAs, and we now have catalogs of over a million such interaction sites that are candidate RNA regulatory elements. However, it is clear that only a small fraction of these elements truly function as regulatory modules, as few show differences in RNA processing when the RBP is knocked down or otherwise altered. As such, large-scale assays to sift through these elements to identify the subset that are function are an essential missing piece in converting these element databases into a useful tool for researchers interested in understanding whether human genetic variation will alter RNA biology. In this proposal, I describe my research group’s proposed efforts to address this major knowledge gap in two areas: 1. Using orthogonal approaches (rapid degradation of RBPs followed by genomics profiling to identify direct regulatory targets of RBPs and massively parallel reporter assays) to characterize which RBP binding sites confer regulation. 2. Large-scale identification of regulatory targets for snoRNAs, snRNAs, and other regulatory RNAs through improved genomics techniques. My extensive expertise in developing experimental and computational genomics methods to map and understand RNA processing regulatory networks makes my newly founded lab an ideal location to undertake these efforts, and build an improved global picture of the RNA processing regulatory landscape. Further, it will support my efforts to develop an independent research group that will lay the foundation to the broader effort to understand how human genetic variation affects disease through mis-regulation of RNA processing.