BPS2026 – The nucleoporin Gle1 stimulates dead-box protein 5 (Dbp5) ATPase and promotes Dbp5-RNA binding to mediate RNA export

The DEAD-box protein Dbp5 is essential for nuclear RNA export and is regulated by a co-factor, Gle1, at the cytoplasmic face of the nuclear pore complex (NPC). Like other characterized DEAD-box proteins, Dbp5 is an RNA-stimulated ATPase. Mechanistic understanding of how Gle1 modulates the Dbp5 ATPase and RNA binding is necessary for understanding the nuclear RNA export pathway. In this work, we report steady-state and transient kinetic and quantitative equilibrium analyses detailing how Gle1 affects the RNA-activated ATPase cycle of Dbp5. Steady-state ATPase assays show that Gle1 lowers the apparent K M for poly-U RNA by almost 300-fold and increases the RNA-activated steady-state cycling rate ( k cat ) ∼2-fold, consistent with previous studies. Global fitting of multiple transient kinetic data sets of RNA, nucleotide, and Gle1 binding favors a pathway in which Gle1 activates Dbp5 by favoring the formation of the ATP-bound state of Dbp5, accelerating rate-limiting P i release, and promoting RNA binding. These observations provide a framework for understanding the role of Gle1 in regulating the Dbp5 ATPase and RNA interactions to mediate RNA export.