eCLASH Towards defining the small RNA interactome

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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.