bioRxiv preprint doi: https://doi.org/10.1101/163998; this version posted July 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Interferon Regulatory Factor 5 (IRF5) is a Regulator of mRNA Translation Interferon Regulatory Factor 5 (IRF5) Interacts with the Translation Initiation Complex and Promotes mRNA Translation During the Integrated Stress Response to Amino Acid Deprivation James D. Warner1,2, Mandi Wiley1, Ying-Y Wu1, Feng Wen1, Michael Kinter3, Akiko Yanagiya4, Kandice L. Tessneer1 and Patrick M. Gaffney1,2 Affiliations: 1Arthritis & Clinical Immunology Research Program; Oklahoma Medical Research Foundation; Oklahoma City, OK, 73104; USA 2Department of Pathology; University of Oklahoma School of Medicine; Oklahoma City, Oklahoma, 73104; USA 3Aging & Metabolism Research Program; Oklahoma Medical Research Foundation; Oklahoma City, OK, 73104; USA 4Department of Biochemistry; Goodman Cancer Research Centre; Montreal, Quebec H3A 1A3; Canada Running Title: Interferon Regulatory Factor 5 (IRF5) is a Regulator of mRNA Translation Corresponding Author: Correspondence and requests for materials should be addressed to Patrick M. Gaffney, Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, MS #57, Oklahoma City, OK 73104, Phone: 405-271-2572, Fax: 405-271-2536, Email: [email protected] Keywords: interferon regulatory factor (IRF), protein synthesis, stress response, unfolded protein response, eukaryotic translation initiation, translation regulation, eukaryotic initiation factor 4G (eIF4G), eukaryotic initiation factor 4E (eIF4E), mRNA, fibroblast ABSTRACT protein expression correlated with increased Interferon Regulatory Factor 5 (IRF5) levels of downstream target genes including plays an important role in limiting pathogenic CHOP and GADD34 and was associated with infection and tumor development. Host amplification of eIF2 de-phosphorylation and protection by IRF5 can occur through a variety translational de-repression under stress. The of mechanisms including production of type I novel mechanism we describe broadens our interferon and cytokines as well as the regulation understanding of how IRF5 regulates gene of cell survival, growth, proliferation, and expression and may govern diverse cellular differentiation. While modulation of these processes in the absence of stimuli that trigger cellular processes is attributed to IRF5 IRF5 nuclear translocation. transcription factor function in the nucleus, emerging evidence suggests that IRF5 may also retain non-transcriptional regulatory properties INTRODUCTION within the cytoplasmic compartment. Consistent Protein synthesis is a process essential with this notion, we report the ability of IRF5 to for cell survival, however it is a function that control gene expression at the level of mRNA renders the cell vulnerable to changes in nutrient translation. Our findings demonstrate that IRF5 availability and viral replication. As a result, interacts with the translation initiation complex safeguards are in place to ensure coordination in the absence of the m7GTP cap-binding between cell activity and environmental protein, eIF4E. We observed that under nutrient conditions. Many of these protective measures deprivation-induced cell stress, IRF5 promoted surround the rate-limiting, initiation step of mRNA translation of the master integrated stress mRNA translation. response (ISR) regulator, Activating During mRNA translation initiation, the Transcription Factor 4 (ATF4). Enhanced ATF4 ribosomal subunits are recruited in stages to the 1 bioRxiv preprint doi: https://doi.org/10.1101/163998; this version posted July 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Interferon Regulatory Factor 5 (IRF5) is a Regulator of mRNA Translation translation start site by eukaryotic initiation including those involved in nutrient uptake and factors (eIFs) that form the translation initiation metabolism, protein folding, and cell survival as complex (TIC). Central to this process is the well as other transcription factors that further m7GTP-binding protein, eIF4E, which binds the regulate a broad array of biological processes 5’-cap of mRNA transcripts; eIF4A, a helicase (15-19). ATF4 also directly promotes that unwinds RNA secondary structure; and upregulation of GADD34, an eIF2 phosphatase eIF4G, a scaffolding protein that binds both that stimulates translational de-repression during eIF4E and eIF4A as well as other factors cell stress and thereby facilitates protein important for mRNA translational regulation synthesis of ISR-related genes (20). including the eIF3 complex (1-4). eIF3 is Protein expression of ATF4 is tightly responsible for TIC recruitment of the 43S pre- regulated and mRNA translation is paradoxically initiation complex which is comprised of the triggered by eIF2phosphorylation (21). An 40S ribosomal subunit, eIF2 bound by GTP, upstream open reading frame (uORF) present and the initiator methionyl-tRNA (Met-tRNAi) within the ATF4 mRNA transcript hinders (5,6). Upon recruitment, 40S ribosomal translation initiation at the ATF4 translation start scanning commences and subsequent site when levels of Met-tRNAi-eIF2-GTP recognition of the start codon by Met-tRNAi (collectively known as ternary complex) are results in eIF2-GTP hydrolysis and its release of abundant (21). However, when less ternary Met-tRNAi as well as departure from the TIC complex is available, prolonged ribosomal along with eIF3 thereby facilitating 60S scanning occurs allowing for uORF bypass and ribosomal recruitment and downstream protein translation of the ATF4 coding sequence (21). synthesis (5,7). IRF5 is a transcription factor that eIF2-GDP can be recycled for additional responds to pathogenic infection, DNA damage, rounds of translation initiation, but only and death receptor (DR) signaling, and therefore following the GDP-for-GTP exchange catalyzed may also be viewed as a regulator of certain by the guanine nucleotide exchange factor, forms of cell stress (22-26). Ligand binding by eIF2B (8). Additionally, four different the pattern recognition receptors, Toll-like mammalian eIF2 kinases (eIF2AKs) exist receptor (TLR) 7 and TLR9, as well as binding which can phosphorylate eIF2 on Ser51 and of DR4/5 by TNF-related Apoptosis-inducing prevent guanine nucleotide exchange resulting in Ligand (TRAIL) leads to downstream signaling inhibition of translation initiation (9,10). These which activates IRF5 (24,27,28). IRF5 eIF2AKs include General Control Non- activation results in dimerization and nuclear derepressible 2 (GCN2), Protein Kinase R translocation where it promotes transcription of (PKR), PKR-like Endoplasmic Reticulum type I interferon (IFN), pro-inflammatory Kinase (PERK), and Heme-Regulated Inhibitor cytokine, and apoptosis-related genes (29,30). (HRI) and each of these kinases act as an While the role of IRF5 in the nucleus is well environmental sensor to different sets of stimuli characterized, a recent report described a (9,10). GCN2 is activated in response to cytoplasmic function of IRF5 unrelated to unbound tRNAs and certain RNA viruses and transcription thereby suggesting that more can therefore provide protection against amino insight is needed into the way IRF5 regulates acid deprivation and infection, respectively, by various cellular processes (31). conserving the supply of amino acids and In support of this idea, we report here a shutting down the translational machinery novel interaction between IRF5 and multiple required for viral replication (11-13). TIC components including eIF4G and eIF4A, In addition to inhibiting the mRNA but not the m7GTP binding protein, eIF4E. translation rate, GCN2 (like other eIF2AKs) Furthermore, we demonstrate that IRF5 upregulates an adaptive gene expression promotes mRNA translation of ATF4 during program known as the integrated stress response amino acid deprivation resulting in enhanced (ISR) (14). Central to the regulation of the ISR expression of ATF4 target genes, protein is the transcription factor, ATF4. ATF4 synthesis, and altered ISR gene signature. Our promotes the transcription of many target genes work provides a broader understanding of the 2 bioRxiv preprint doi: https://doi.org/10.1101/163998; this version posted July 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Interferon Regulatory Factor 5 (IRF5) is a Regulator of mRNA Translation way in which IRF5 influences cell stress combination of a 30-base pair insertion/deletion responses. (indel) polymorphism and an alternative splice site that results in loss of the first 48 base pairs RESULTS of exon 6 (Fig. S2a) (35). To investigate IRF5 interacts with the translation whether structural variability in IRF5 would initiation complex independently of eIF4E— impact IRF5 association with the TIC, we co- Mass spectrometry analyses of IRF5 expressed HA-tagged eIF4G with four of the immunoprecipitation reactions suggested most common FLAG-tagged IRF5 variants (V1, associations between IRF5 and several RNA- V2/V6, V3/V4, and V5) in HEK293T cells (Fig. binding proteins implicated in mRNA translation S2b). The scaffolding protein, eIF4G, which including 40S ribosomal proteins, translation