Two-Site Phosphorylation of EPRS Coordinates Multimodal Regulation of Noncanonical Translational Control Activity

Abstract

Summary Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric GAIT (Gamma-interferon Activated Inhibitor of Translation) complex that binds 3′UTR GAIT elements in multiple interferon-gamma (IFN-γ)-inducible mRNAs and suppresses their translation. Here we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-γ induces sequential phosphorylation of Ser886 and Ser999 in the non-catalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser886 phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH, and restores mRNA binding. Finally, Ser999 phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-site phosphorylation provides structural and functional pliability to EPRS, and choreographs the repertoire of activities that regulates inflammatory gene expression.

A versatile assay for RNA-binding proteins in living cells

“…However, in a biological context the RNAbinding activity of RBPs is typically regulated by a plethora of mechanisms, including post-translational modifications (Mukhopadhyay et al 2008;Arif et al 2009), induced conformational changes (Walden et al 2006;Siddiqui et al 2012), competition with other RBPs for target RNAs (Yao et al 2012), differential subcellular localization (Alvarez et al 2013), alterations of its expression levels, or the availability of its target RNAs. Importantly, most of the proteins within the HeLa mRNA interactome are post-translationally modified (http://www.embl.de/mRNAinteractome) (Castello et al 2012), suggesting that RBPs may globally respond to environmental alterations.…”

“…As an illustrative example, glutamyl-prolyl tRNA synthetase (EPRS) is phosphorylated in macrophages in response to interferon-γ treatment, triggering its release from the multi-synthetase complex and its subsequent assembly into the IFN-γ-activated inhibitor of translation (GAIT) complex ). Phosphorylation also promotes conformational changes in the WHEP domains of EPRS that activate its mRNA-binding activity, enabling translational repression of proinflammatory genes (Jia et al 2008;Arif et al 2009Arif et al , 2011. The REM (RNA, enzyme, and metabolite) network hypothesis (Hentze and Preiss 2010) proposes an additional layer of RNA-binding regulation, where substrate (or cofactor) and RNA compete for the metabolitebinding pocket of an enzyme.…”

Heterotrimeric GAIT Complex Drives Transcript-Selective Translation Inhibition in Murine Macrophages

“…The human GAIT complex is assembled in two distinct stages. After about 2 to 4 h of IFN-␥ treatment, EPRS is phosphorylated at two sites, first at Ser 886 by cyclin-dependent kinase 5 (Cdk5) and subsequently at Ser 999 by a Cdk5-dependent AGC kinase (2,3). Both phosphorylation events occur in the noncatalytic linker domain that joins the functional ERS and PRS synthetases.…”

A truncated tRNA synthetase directs a “translational trickle” of gene expression

“…1,2 In human myeloid cells, IFNγ induces phosphorylation of EPRS at two Ser residues in the noncatalytic linker domain connecting the synthetase cores. 3 Both phosphorylation events are required for recruitment of other GAIT complex proteins, including L13a, which binds initiation factor eIF4G and blocks recruitment of the pre-initiation complex, thereby inhibiting translation of GAIT target mRNAs. 1,3 Our recent discovery of a new level of regulation emerged from a detailed analysis of an IFNγ dose-response experiment.…”

“…3 Both phosphorylation events are required for recruitment of other GAIT complex proteins, including L13a, which binds initiation factor eIF4G and blocks recruitment of the pre-initiation complex, thereby inhibiting translation of GAIT target mRNAs. 1,3 Our recent discovery of a new level of regulation emerged from a detailed analysis of an IFNγ dose-response experiment. Following 8-h treatment of cells with a range of IFNγ concentrations, we observed a linear relationship between VEGF-A mRNA and VEGF-A protein in lysates.…”