Single molecule fluorescence approaches reveal that DEAD-Box RNA helicases cluster in dynamic hubs for protein synthesis initiation
Abstract
DEAD-box helicases, DDX3 and eukaryotic initiation factor 4A (eIF4A), play essential roles in translation initiation, cytoplasmic surveillance for foreign RNAs and in facilitating cellular stress response. Using multi-parameter confocal fluorescence burst spectroscopy, we discovered that sex chromosome-encoded helicases, DDX3X and DDX3Y, form 20 nm RNA-protein clusters (RPCs) containing a few RNA duplexes and dozens of protein subunits. This assembly occurs at nanomolar concentrations, far below the critical micromolar concentration threshold for liquid-liquid phase separation. N-terminal and C-terminal intrinsically disordered regions (IDRs) are required for RPC formation of DDX3s. eIF4A, with much smaller IDRs, does not form RPCs without other protein partners. But with cofactors eIF4B, which is highly disordered, and eIF4G, a scaffolding factor needed to recognize and activate authentic capped mRNAs, the RPCs appear and again facilitate the helicase (unwinding) function. A mutation in the RNA-binding module of eIF4B causes reduced RPC formation, reduced phase separation into condensates, and reduced activity. Faster diffusion of the mutant eIF4B in HeLa cells relative to wild type implies that these features apply in cells as well as in vitro. As DEAD-Box helicases are not processive, RPCs may be required for unwinding of larger mRNA secondary structures during initiation of protein synthesis and in formation of stress granules as a cellular protective mechanism.
Biography
Dr. Goldman obtained a BS in Electrical Engineering from Northwestern University in 1969 and MD and PhD in Physiology from the University of Pennsylvania in 1975. He was Post-Doctoral Fellow at University College London under Professor Sir Andrew F. Huxley and Robert M Simmons until 1980 at which time he joined the Physiology Department of the School of Medicine, University of Pennsylvania. He moved his laboratory and appointment to University of California in Davis in 2023. Dr. Goldman has developed novel instrumentation for biophysical studies on muscle contraction, non-muscle molecular motors, protein synthesis and RNA helicases. He introduced photochemical approaches to transient dynamics and mechanochemistry of these biophysical systems, including caged ATP photolysis, high speed optical traps (laser tweezers), stable isotope oxygen exchange, single molecule nanometer tracking and polarized total internal reflection fluorescence microscopy. He was awarded the Bowditch Lectureship of the American Physiological Society, Kinosita Single Molecule Award, President, and Fellow of the Biophysical Society, Storer Lectureship at UC Davis among others. He is a Fellow of the American Academy of Arts and Sciences, American Association for the Advancement of Science and elected Member of the US National Academy of Science.
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