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 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 10-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 nonstressed 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.
Highlights
Throughout the gene expression pathway, the fate of a protein-coding mRNA transcript is determined by interactions between its specific complement of RNA-binding proteins and other cellular machinery
Human Gle1’s N-terminal region contains a much longer intrinsically disordered region (IDR) and is the site of dynamic, stress-induced phosphorylation that alters the formation of Gle1 oligomers in vitro [16], suggesting that other regions of the N terminus contribute to oligomerization
Our prior studies show that perturbation of the predicted Nterminal coiled-coil domain by the lethal congenital contracture syndrome 1–linked PFQ insertion impedes gle1-Finmajor oligomerization and nucleocytoplasmic shuttling, as well as poly(A)1 RNA export [7]
Summary
Throughout the gene expression pathway, the fate of a protein-coding mRNA transcript is determined by interactions between its specific complement of RNA-binding proteins and other cellular machinery. Because expression of Gle1152–360, containing only the coiled-coil domain, did not produce high-molecular-weight oligomers, we speculated that a different region in the N-terminal region of Gle1 mediates the disc formation. These results demonstrated that the aggregation-prone region of amino acids 45–54 is necessary but not sufficient in combination with the coiled-coil domain to drive higher-ordered Gle1 oligomerization.
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