Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Thursday Speaker Abstracts

NANOMETER-SCALE RNA PROTEIN CLUSTERS (RPCS) FOSTER HELICASE ACTIVITY OF DEAD-BOX HELICASE EIF4A Yale E. Goldman 1,2 ; Him Shweta 1,2 ; Masaaki Sokabe 2 ; Christopher S Fraser 2 ; 1 University of California, Davis, Department of Pharmacology, Davis, CA, USA 2 University of California, Davis, Department of Molecular and Cellular Biology, Davis, CA, USA DEAD-box RNA helicases are central regulators of RNA metabolism, employing ATP dependent mechanisms to remodel RNA structure and RNA-protein interactions. The translation initiation helicase, eukaryotic initiation factor 4A (eIF4A), is a non-processive enzyme essential for unwinding structured mRNAs that relies on cofactors to achieve physiological activity. Using a single molecule approach we uncovered an unexpected RNA-helicase state of eIF4A that forms into nanometer-scale RNA-protein clusters (RPCs) of ~2-5 MDa in presence of its physiological cofactors eIF4B and eIF4G, RNA and ATP under near-physiological concentrations. Discrete clusters recruit multiple copies of protein subunits with RNA upon ATP addition. RPC formation correlates with helicase activity in vitro. eIF4B is a key determinant of this multi-subunit assembly. Its intrinsically disordered regions (IDRs) together with structured RNA-recognition motifs (RRMs) drive multivalent RNA-dependent interactions, critical for efficient helicase activity. Disrupting eIF4B-RNA interactions through a targeted point mutation (F139A) in the RRM reduces both the cluster size and the helicase activity, further establishing the functional link between cluster formation and catalytic activity. In-cell diffusion measurements reveal markedly slower diffusion of wild-type eIF4B compared with the RNA-binding-deficient mutant, indicative of RPC formation also in the cellular environment. Together, our results reveal regulated helicase clustering as a previously unrecognized characteristic of the translation initiation machinery, linking ATP-dependent DEAD-box helicase activity to nanometer-scale RNA-protein clusters and translation initiation regulation.

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