RNA helicases DDX3X and DDX3Y form nanometer-scale RNA-protein clusters that support catalytic activity

Amber Yanas; Him Shweta; Michael C Owens; Kathy Fange Liu; Yale E Goldman

doi:10.1016/j.cub.2024.10.055

RNA helicases DDX3X and DDX3Y form nanometer-scale RNA-protein clusters that support catalytic activity

Curr Biol. 2024 Nov 21:S0960-9822(24)01449-0. doi: 10.1016/j.cub.2024.10.055. Online ahead of print.

Authors

Amber Yanas¹, Him Shweta², Michael C Owens¹, Kathy Fange Liu³, Yale E Goldman⁴

Affiliations

¹ Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
² Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
³ Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn Institute for RNA Innovation, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn Center for Genome Integrity, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: liufg@pennmedicine.upenn.edu.
⁴ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: yegoldman@ucdavis.edu.

PMID: 39591970
DOI: 10.1016/j.cub.2024.10.055

Abstract

DEAD-box helicases, crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs) whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nanometer-scale RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single-molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. The N-terminal IDRs of DDX3X and DDX3Y are both the drivers of RPC formation and the major differentiators of their enzymatic activities. Our findings provide new insights that the nanoscale helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and might act as nucleation points for stress granule formation. This mechanism may apply broadly among other members of the DEAD-box helicase family.

Keywords: DEAD-box; FRET; RNA; cancer; dcFCCS; helicase; sex chromosomes; single molecule; translation initiation; unwinding.