The GAIT translational control system

Abstract

The interferon (IFN)-γ-activated inhibitor of translation (GAIT) system directs transcript-selective translational control of functionally related genes. In myeloid cells, IFN-γ induces formation of a multiprotein GAIT complex that binds structural GAIT elements in the 3'-untranslated regions (UTRs) of multiple inflammation-related mRNAs, including ceruloplasmin and VEGF-A, and represses their translation. The human GAIT complex is a heterotetramer containing glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a (L13a), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A network of IFN-γ-stimulated kinases regulates recruitment and assembly of GAIT complex constituents. Activation of cyclin-dependent kinase 5 (Cdk5), mammalian target of rapamycin complex 1 (mTORC1), and S6K1 kinases induces EPRS release from its parental multiaminoacyl tRNA synthetase complex to join NSAP1 in a 'pre-GAIT' complex. Subsequently, the DAPK-ZIPK kinase axis phosphorylates L13a, inducing release from the 60S ribosomal subunit and binding to GAPDH. The subcomplexes join to form the functional GAIT complex. Each constituent has a distinct role in the GAIT system. EPRS binds the GAIT element in target mRNAs, NSAP1 negatively regulates mRNA binding, L13a binds eIF4G to block ribosome recruitment, and GAPDH shields L13a from proteasomal degradation. The GAIT system is susceptible to genetic and condition-specific regulation. An N-terminus EPRS truncate is a dominant-negative inhibitor ensuring a 'translational trickle' of target transcripts. Also, hypoxia and oxidatively modified lipoproteins regulate GAIT activity. Mouse models exhibiting absent or genetically modified GAIT complex constituents are beginning to elucidate the physiological role of the GAIT system, particularly in the resolution of chronic inflammation. Finally, GAIT-like systems in proto-chordates suggests an evolutionarily conserved role of the pathway in innate immunity. WIREs RNA 2018, 9:e1441. doi: 10.1002/wrna.1441 This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Riboswitches.

Figure 1

Schematic diagram depicting human interferon γ‐activated inhibitor of translation (GAIT) system. Interferon‐γ (IFN‐γ) induces formation of heterotetrameric GAIT complex in myeloid cells. The human GAIT complex contains glutamyl‐prolyl tRNA synthetase (EPRS), NS1‐associated protein 1 (NSAP1), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and ribosomal protein (RP) L13a. Murine GAIT complex is essentially identical but lacks NSAP1.

Figure 2

Schematic diagram of interferon γ‐activated inhibitor of translation (GAIT) system activation for transcript‐selective translation inhibition of inflammation‐related genes in myeloid cells. Interferon‐γ (IFN‐γ) induces early activation of human EPRS by two‐step phosphorylation at Ser⁸⁸⁶ and Ser⁹⁹⁹ in the linker region by Cdk5/p35, mTORC1, and S6K1 kinases. Cdk5/p35 directly phosphorylates Ser⁸⁸⁶, and is also required, in conjunction with mTORC1 and S6K1 for phosphorylation of Ser⁹⁹⁹. Phosphorylated‐EPRS (P‐EPRS) is released from the multiaminoacyl tRNA synthetase complex (MSC). Phospho‐Ser⁸⁸⁶ EPRS interacts with NSAP1 to form an inactive pre‐GAIT complex (in humans only). After ~12–16 h, L13a is phosphorylated at Ser⁷⁷ (P‐L13a) by DAPK‐activated ZIPK, and released from the 60S ribosomal subunit. Free P‐L13a interacts with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and joins the pre‐GAIT complex to form the functional GAIT complex. The complex binds GAIT elements in the 3′‐UTR of functionally related transcripts induced by IFN‐γ, and inhibits their translation by a mechanism that requires a circularized mRNA. L13a in the GAIT complex interferes with the eukaryotic translation‐initiation factor, eIF4G, near the eIF3‐binding site, and blocks translation‐initiation. DAPK and ZIPK translations are repressed by GAIT system constituting a negative‐feedback module to limit L13a phosphorylation and GAIT system activity.

Figure 3

Summary of canonical and noncanonical activities of interferon γ‐activated inhibitor of translation (GAIT) complex constituent proteins.

Figure 4

Schematic diagram of glutamyl‐prolyl tRNA synthetase (EPRS) domains, phospho‐sites and their interactors. An N‐terminal glutathione‐S‐transferase (GST)‐like domain is upstream of the ERS domain, which is joined to the PRS domain by a linker containing three WHEP repeats: R1, R2, and R3. In humans, NSAP1 binding to EPRS in the R2–R3 region requires Ser⁸⁸⁶ phosphorylation. Phospho‐Ser⁷⁷‐L13a and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) interaction with EPRS requires Ser⁹⁹⁹ phosphorylation. The interferon γ‐activated inhibitor of translation (GAIT) element RNA binds in the upstream R1–R2 repeat region.

Figure 5

Sequence and structure characteristics of a typical bipartite stem‐loop interferon γ‐activated inhibitor of translation (GAIT) element with conserved nucleotides in bold (left), and examples of validated functional GAIT elements (right). The structure is derived from the biochemical and mutagenesis experiments on human 3′‐UTR Cp GAIT element.

Figure 6

Regulation of interferon γ‐activated inhibitor of translation (GAIT) system in normoxic and hypoxic macrophages, and control of GAIT element‐bearing VEGFA mRNA. (i) The GAIT system is activated by IFN‐γ‐dependent phosphorylation of EPRS and L13a in myeloid cells. (ii) Stimulation with both IFN‐γ and oxidatively modified low density lipoprotein, LDL (LDL_ox) induces activation of an iNOS‐S100A8/A9 nitrosylase complex and site‐specific S‐nitrosylation of an array of targets bearing an I/L‐X‐C‐X₂‐D/E motif, including Cys²⁴⁷ of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). SNO‐GAPDH fails to bind L13a causing proteasomal degradation of the latter and inactivation of the GAIT system. (iii) In hypoxia, IFN‐γ‐induced hnRNP L is phosphorylated at Tyr³⁵⁹ and joins hnRNP A2/B1 and double‐stranded RNA‐binding protein (RBP), DRBP76, to form the active HILDA complex. The complex binds the hypoxia stability region (HSR) in VEGFA mRNA, blocks GAIT element conformation, and switches to translation‐permissive conformation. (iv) In normoxia, translational repression of IFN‐γ‐induced VEGFA mRNA by the GAIT complex is facilitated by degradation of hnRNP L following prolyl hydroxylation and von Hippel‐Lindau (VHL)‐mediated polyubiquitination. (v) Constitutive generation of an EPRS truncate, EPRS^N1 by polyadenylation‐directed conversion of a Tyr to a stop codon (PAY*) binds GAIT elements in target mRNAs including VEGFA and shields it from the GAIT complex, thereby allowing a ‘translational trickle’ of expression.