SPIDR: a novel and highly multiplexed method for mapping RNA-protein interactions to comprehensively study post-transcriptional gene expression regulation
Assistant Professor, Biological Sciences, Columbia University, NY
October 31, 2022 | 3:30 PM
Ammon-Pinizzotto Biopharmaceutical Innovation (BPI) Building
Conference Room 140
Characterizing RNA-protein interactions is central to understanding post-transcriptional gene expression regulation. One of the primary methods for studying direct RNA-protein interactions is UV crosslinking and immunoprecipitation (CLIP) coupled with high-throughput sequencing. While powerful, this approach is limited to studying a single RNA-binding protein (RBP) at a time and requires a large number of cells (e.g. 20 million). Consequently, this hinders the study of large classes of RBPs in your system of interest, and especially in rare cell populations and disease-relevant patient samples. However, because these binding maps are highly specific for individual cell types, there is a critical need to enable the generation of comprehensive RNA-protein interaction maps for any cell type of interest. Here we present SPIDR (Split and Pool Immunoprecipitation for Detection of RNPs), a highly multiplexed method to simultaneously profile the global RNA-binding sites of dozens to hundreds of RBPs in a single experiment, thereby increasing the throughput of current methods by two orders of magnitude. SPIDR works by coupling unique barcodes to protein G beads, each bound to an antibody against a protein of interest. This pool of barcoded beads is used to perform CLIP on a single sample, followed by split-and-pool tagging to resolve individual RNA-protein interactions. Importantly, profiling up to a hundred of RBPs within a single experiment has the potential to eliminate batch effects and dramatically reduces time and resources needed. Moreover, SPIDR allows for mapping of multiple protein-RNA interactions in several samples simultaneously, which provides the means for direct comparison of RNA-protein interactions between different samples with minimal interference of systematic errors. SPIDR thus has the potential to enable rapid, de novo discovery of RNA-protein interactions and to comprehensively define RNA-binding sites across multiple cell types – for known and novel RBPs – at an unprecedented scale.
Biography:
Marko Jovanovic did his PhD in the lab of Michael Hengartner at the University of Zurich, Switzerland. In close collaboration with the Ruedi Aebersold lab at the ETH Zurich, they developed novel large-scale approaches to identify microRNA targets genes in C. elegans. After his PhD, he joined the group of Aviv Regev at the Broad Institute of MIT and Harvard in Cambridge, USA. In collaboration with the Steven Carr lab at the Broad Institute, they developed new methods to integrate transcriptomics and proteomics data in order to gain new insight about how protein level changes are regulated during the immune response of dendritic cells. Marko started his own research group at Columbia University in 2017. The overarching research goal of his lab is to understand the principles and mechanisms by which translational regulation controls the dynamics of gene expression and therefore affects processes like differentiation, stress response and pathogenesis.