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DTSTART;TZID=America/Detroit:20220926T160000
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SUMMARY:RNA Innovation Seminar: David Shechner\, University of Washington
DESCRIPTION:“Meet the neighbors: A universal technology for probing RNA-interactions and RNA-scaffolded subcellular compartments in situ”\nDavid Shechner Ph.D.\nAssistant Professor of Pharmacology\nUniversity of Washington \n  \nFlyer in PDF \nIn-person: BSRB\, ABC seminar rooms / hybrid link \nKeywords: RNA\, proximity-biotinylation\, subcellular architecture\, nuclear architecture\, spatial biology\, biomolecular condensates\, interactomics\, nucleolus\, Xist. \n  \nAbstract: In the context of the living cell\, very little RNA is naked. RNA molecules form complex\, dynamic networks of molecular interactions that underlie a host of biochemical functions\, and which are central to organizing subcellular compartmentalization. In humans\, for example\, RNAs are key determinants of chromatin folding\, and they nucleate and scaffold a host of biomolecular condensates that collectively control cellular metabolic\, epigenetic\, and stress-signaling pathways. But\, characterizing these structures—identifying the biomolecules within an RNA’s subcellular microenvironment—remains technically cumbersome. \nTo address this challenge\, I introduce oligonucleotide-mediated proximity-interactome mapping (O-MAP)\, a straightforward and flexible method for identifying the proteins\, RNAs\, and genomic loci near a target RNA\, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA. These enzymes then pervasively label all nearby (~20 nm) molecules\, enabling their enrichment by streptavidin pulldown. O-MAP induces exceptionally precise RNA-targeted biotinylation\, and its modular design enables straightforward validation of probe pools and real-space optimization of the biotinylation radius\, thus overcoming key technical challenges for the field. Moreover\, O-MAP can be readily ported across different target RNAs and specimen types\, including patient-derived organoids and tissue samples. And\, O-MAP achieves this without complex cell-line engineering\, using only off-the-shelf parts and standard manipulations. \nUsing a small cohort of model RNAs\, we have developed a robust O-MAP toolkit for proteomic (O-MAP-MS)\, transcriptomic (O-MAP-Seq) and genome interaction (O-MAP-ChIP) discovery. O-MAP of the 47S-pre-rRNA—the long noncoding RNA that scaffolds the nucleolus—enabled a comprehensive “multi-omic” analysis of this subnuclear structure\, and revealed hundreds of novel nucleolar protein-\, RNA-\, and chromatin interactions. O-MAP of XIST—the master regulator of X-chromosome inactivation—revealed novel RNAs that may play a role in this process\, and unanticipated interactions between XIST and other chromatin-regulatory RNAs. Finally\, targeting O-MAP to introns within a key cardiac developmental gene enabled unprecedented molecular dissection of a subnuclear compartment that would be impossible to purify biochemically. \nGiven these results\, we believe that O-MAP will be a powerful tool for elucidating the mechanisms by which RNA molecules drive subcellular compartmentalization in time and space\, with particular impact on our understanding of nuclear architecture. Moreover\, with O-MAP’s precision\, flexibility\, and ease\, we anticipate its broad use in studying countless other RNA phenomena throughout biology\, and as a clinical diagnostic- and discovery tool.
URL:https://rna.umich.edu/events/david-shechner/
CATEGORIES:Seminar
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