eCLASH Towards defining the small RNA interactome

在要描述的第一类非编码RNA中，微小RNA（miRNA）代表了广泛的、组织特异性的转录后调节系统，其在几乎每种细胞类型中都有活性。miRNAs直接靶向mRNA进行转录后抑制，对生物体的复杂性、发育和功能至关重要。异常的miRNA表达与癌症、代谢疾病和神经退行性变等不同的疾病状态有因果关系。理解miRNA生物学的唯一最大挑战是我们无法明确预测或确定mRNA靶向，目前的预测方法在靶点预测中提供约20%的准确性。在这里，我们建立在该领域的技术进步和目前的原型技术，eCLASH的数据，能够公正的直接确定基本上所有的miRNA-mRNA靶相互作用事件。在这个项目中，我们建议开发和验证该技术，以生成可用于新鲜或冷冻（生物库）原代组织和细胞类型的普遍适用的方案。由于我们正在使用高通量技术，该项目的一个重要部分是建立一个强大的生物信息学管道，能够在生物学家友好的平台上处理高度复杂的数据集。最后，我们的目标是利用这项技术来产生一个强大的新社区资源，在小鼠的至少25个关键组织中绘制所有miRNA-mRNA相互作用事件。重要的是，在这个小鼠相互作用组图谱的背景下，我们将直接绘制对照和代谢性疾病（Db/Db）小鼠的全深度miRNA相互作用谱，从而为eCLASH在理解疾病相关的miRNA依赖性基因调控中的实用性提供概念验证。

Amongst the first classes of non-coding RNAs to be described, microRNAs (miRNAs) represent a broad, tissue-specific post-transcriptional regulatory system active in virtually every cell-type. MiRNAs directly target mRNAs for post-transcriptional repression and are vital to organism complexity, development, and function. Aberrant miRNA expression has been causally implicated in disease states as disparate as cancer, metabolic disease, and neurodegeneration. The single greatest contemporary challenge for understanding miRNA biology is our inability to definitively predict or determine mRNA targeting, with current predictive methods providing ~20% accuracy in target prediction. Here, we build upon technological advances in the field and present data for a prototype technology, eCLASH, capable of unbiased direct determination of essentially all miRNA-mRNA target interaction events. In this project, we propose development and validation of the technology to the point of generating a universally applicable protocol usable in fresh or frozen (biobanked) primary tissues and cell-types. Since we are working with a high-throughput technology, an important part of the project is to establish a robust bioinformatics pipeline able to tackle the highly complex datasets in a biologist friendly platform. Finally, we aim to harness the technology to generate a powerful new community resource, mapping all miRNA-mRNA interaction events in at least 25 key tissues of the mouse. Critically, within the context of this mouse interactome atlas, we will directly map the full depth miRNA interaction profiles of control and metabolically diseased (Db/Db) mice and thus provide proof-of-concept for the utility of eCLASH in understanding disease associated miRNA-dependent gene regulation.