Transcriptome-wide nuclease-mediated protein footprinting to identify RNA-protein interaction sites. I. Silverman1,2, F. Li1, Q. Zheng1, B. Gregory1,2 1) Department of Biology; 2) Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA.

   RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, investigating the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions focus on individual RBPs. However, there are hundreds of these proteins encoded in the human genome, and each cell type expresses a different repertoire, greatly limiting the ability of current methods to capture the global landscape of RNA-protein interactions. To address this gap, we and others have recently developed methods to globally identify regions of RNAs that are bound by proteins in an unbiased manner. Here, we present our ribonuclease-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). We describe the application and validation of this protocol in multiple mammalian cell lines. We identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites. Finally, we use structure-specific nuclease digestion patterns generated by these methods to reveal the local RNA secondary structure at binding sites for several RBPs, including the double-stranded RNA binding protein and component of the microRNA processing machinery, DGCR8. Intriguingly our results suggest that highly structured regions of human mRNAs are targeted by the microprocessor complex, resulting in endonucleolytic cleavage and production of functional small RNAs. Our results offer insights into global patterns of RNA-protein interactions, reveal the structural contexts of RBP binding sites, and uncover a novel mechanism for microRNA machinery-mediated regulation. Future applications of this method to study the dynamics of RNA-protein interactions and RNA secondary structure in developmental and disease processes will help to uncover the role of RBPs in post-transcriptional regulation.

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