MicroRNAs (miRNAs) are a class of small, non-coding RNA that play a key role in post-transcriptional regulation of gene expression via translational suppression and mRNA degradation. miRNAs are predicted to be responsible for the regulation of greater than 60% of human protein-coding genes. Accordingly, the generation, abundance, and function of a miRNA is tightly regulated, both transcriptionally and post-transcriptionally. Post-transcriptional regulation of miRNA processing, abundance, and function is mediated by auxiliary RNA binding proteins (RBPs), which occurs in many cases through binding the terminal loop of precursor miRNA species. However, the specific RBPs that regulate most miRNAs are unknown. Current methods for comprehensively profiling the RNA-protein interactions are not applicable to studying the pre-miRNA-protein interactome. Furthermore, subsequent validation of putative pre-miRNA-protein interactions remains challenging due to the technical requirements of existing biochemical and cellular methods used to detect RNA-protein interactions. To address gaps in current technologies, I am developing methods for the discovery and validation of cellular pre-miRNA-protein interactions. To enable the identification of proteins that interact with a specific pre-miRNA sequence of interest, I am optimizing an RNA-centric proteomics-mediated assay aided by photoactivatable ribonucleoside-enhanced crosslinking and biotin-streptavidin affinity purification. Additionally, I am expanding a cellular assay, under development in the Garner laboratory, for detection of pre-miRNA-RBP interactions, termed RNA interaction with Protein-mediated Complementation Assay (RiPCA), which integrates Promega’s HaloTag and NanoLuc technology to detect direct pre-miRNA-RBP interactions. I have recently completed optimization experiments using well-validated pre-let-7/Lin28 interactions as proof-of-concept. Further development of this method is focused on expanding RiPCA to enable the detection of small molecule-mediated pre-miRNA-RBP interaction inhibition, as well as other pre-miRNA-RBP interactions. Together, these new methods of discovering and validating pre-miRNA-protein interactions will facilitate the study of miRNA-RBP interaction networks.

Developing methods for the discovery and validation of pre-miRNA-protein interactions

2019 Fall National Meeting

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