RNA-protein interactions are integral to the regulation of gene expression. RNAs have diverse functions and the protein interactomes of individual RNAs vary temporally, spatially, and with physiological context. These factors make the global acquisition of individual RNA-protein interactomes an essential endeavor. Although techniques have been reported for discovery of the protein interactomes of specific RNAs they are largely laborious, costly, and accomplished singly in individual experiments. We developed HyPR-MS for the discovery and analysis of the protein interactomes of multiple RNAs in a single experiment while also reducing design time and improving efficiencies. Presented here is the application of HyPR-MS to simultaneously and selectively isolate the interactomes of lncRNAs MALAT1, NEAT1, and NORAD. Our analysis features the proteins that potentially contribute to both known and previously undiscovered roles of each lncRNA. This platform provides a powerful new multiplexing tool for the efficient and cost-effective elucidation of specific RNA-protein interactomes.
Proteins bind mRNA through their entire life cycle from transcription to degradation. We analyzed c-Myc mRNA protein interactors in vivo using the HyPR-MS method to capture the crosslinked mRNA by hybridization and then analyzed the bound proteins using mass spectrometry proteomics. Using HyPR-MS, 229 c-Myc mRNA-binding proteins were identified, confirming previously proposed interactors, suggesting new interactors, and providing information related to the roles and pathways known to involve c-Myc. We performed structural and functional analysis of these proteins and validated our findings with a combination of RIP-qPCR experiments, in vitro results released in past studies, publicly available RIP-and eCLIP-seq data, and results from software tools for predicting RNA-protein interactions.
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