Functions of Gle1 are governed by two distinct modes of self-association

Abstract

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate (IP6) binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel ten amino acid aggregation prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and non-stressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and promote Dbp5-mediated tRNA export in vivo in Saccharomyces cerevisiae

“…Following ssRNA binding, hydrolysis of ATP to ADP results in a reduction in the affinity of Dbp5 for the ssRNA substrate ( Weirich et al, 2006 ). While there has been extensive characterization of the structural and biochemical details of Dbp5, ssRNA, and co-regulators ( Wong et al, 2018 ; Mason and Wente, 2020 ; Gray et al, 2022 ), no such understanding exists for Dbp5 engaging highly structured RNA substrates like tRNA. As such, it was first tested if recombinant full-length Dbp5 could bind commercially available yeast mixed tRNAs or the yeast phenylalanine tRNA (both substrates that have been used in previous biochemical and structural studies; Shi and Moore, 2000 ; Yao et al, 2007 ).…”

A New Phase of Networking: The Molecular Composition and Regulatory Dynamics of Mammalian Stress Granules

“…Other experiments reveal more profound influences of phosphorylation on stress granule formation. For example, hyperphosphorylation of GLE1A isoforms by JNK, ERK, and GSK3 during stress recovery impairs its oligomerization and ability to modulate the ATPase activity of the DEAD-box helicase DDX3X, subsequently promoting SG disassembly. , Similarly, the dissolution of SGs following acute heat stress is reliant upon phosphorylation of the ATPase VCP by the serine/threonine protein kinases ULK1/ULK2 . Finally, phosphorylation on G3BP1 affects SG dynamics, a point recently clarified after a long controversy.…”

Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and to promote Dbp5 mediated tRNA export in vivo

“…Following ssRNA binding, hydrolysis of ATP to ADP results in a reduction in the affinity of Dbp5 for the ssRNA substrate (Weirich, Erzberger et al 2006 ). While there has been extensive characterization of the structural and biochemical details of Dbp5, ssRNA, and co-regulators (Wong, Gray et al 2018, Mason and Wente 2020, Gray, Cao et al 2022 ), no such understanding exists for Dbp5 engaging highly structured RNA substrates like tRNA. As such, it was first tested if recombinant full length Dbp5 could bind commercially available yeast mixed tRNAs or the yeast Phenylalanine tRNA (both substrates that have been used in previous biochemical and structural studies (Shi andMoore 2000 , Yao, Roser et al 2007 )).…”