Poster abstracts
Poster number 57 submitted by Jackson Trotman
Protein interactome analysis reveals stabilization of mammalian capping enzyme by heat shock protein 90
Jackson B. Trotman (Department of Biological Chemistry and Pharmacology, The Ohio State University), Bernice Agana (Department of Chemistry and Biochemistry, The Ohio State University), Andrew J. Giltmier (Department of Biological Chemistry and Pharmacology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Daniel R. Schoenberg (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
A defining feature of eukaryotic messenger RNAs (mRNAs) is the presence of a 5′ N7-methylguanosine cap, which is essential for proper RNA processing, localization, and translation. Beyond the canonical nuclear cap synthesis pathway, mammalian cells also possess cytoplasmic machinery capable of restoring a cap structure onto uncapped and/or partially degraded RNA 5′ ends. Central to both pathways is capping enzyme (CE), a bifunctional triphosphatase-guanylyltransferase that localizes to both the nucleus and the cytoplasm. To gain broader insight into the cellular context of cytoplasmic recapping, we sought to characterize the protein interactome of cytoplasmic CE (cCE). Stable U2OS cell lines were generated to inducibly express a tagged form of cCE or negative control EGFP, and these bait proteins were affinity purified and analyzed by bottom-up proteomics to identify interacting partners. Several cCE-interacting proteins with broad cellular functions were identified and validated, including the 90-kDa heat shock protein (HSP90). Specifically blocking HSP90 function with three different inhibitory drugs (geldanamycin, radicicol, and onalespib) caused levels of both nuclear and cytoplasmic CE to destabilize, establishing CE as a novel HSP90 client protein.
Keywords: capping enzyme, proteomics, HSP90