eIF3d controls the persistent integrated stress response.

Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.

Convergent mutations in phage virion assembly proteins enable evasion of Type I CBASS immunity.

CBASS is an anti-phage defense system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by viral DNA but the stage of phage replication which activates bacterial CBASS remains unclear. Here we define the specificity of Type I CBASS immunity using a comprehensive analysis of 975 operon-phage pairings and show that Type I CBASS operons composed of distinct CD-NTases, and Cap effectors exhibit striking patterns of defense against dsDNA phages across five diverse viral families. We demonstrate that escaper phages evade CBASS immunity by acquiring mutations in structural genes encoding the prohead protease, capsid, and tail fiber proteins. Acquired CBASS resistance is highly operon-specific and typically does not affect overall fitness. However, we observe that some resistance mutations drastically alter phage infection kinetics. Our results define late-stage virus assembly as a critical determinant of CBASS immune activation and evasion by phages.

Moonlighting translation factors: multifunctionality drives diverse gene regulation.

Translation factors have traditionally been viewed as proteins that drive ribosome function and ensure accurate mRNA translation. Recent discoveries have highlighted that these factors can also moonlight in gene regulation, but through functions distinct from their canonical roles in protein synthesis. Notably, the additional functions that translation factors encode are diverse, ranging from transcriptional control and extracellular signaling to RNA binding, and are highly regulated in response to external cues and the intrinsic cellular state. Thus, this multifunctionality of translation factors provides an additional mechanism for exquisite control of gene expression.

Eukaryotic Initiation Factor 5A2 Regulates Expression of Antiviral Genes.

Translation factors are essential for regulation of protein synthesis. The eukaryotic translation initiation factor 5A (eIF5A) family is made up of two paralogues - eIF5A1 and eIF5A2 - which display high sequence homology but distinct tissue tropism. While eIF5A1 directly binds to the ribosome and regulates translation initiation, elongation, and termination, the molecular function of eIF5A2 remains poorly understood. Here, we engineer an eIF5A2 knockout allele in the SW480 colon cancer cell line. Using ribosome profiling and RNA-Sequencing, we reveal that eIF5A2 is functionally distinct from eIF5A1 and does not regulate transcript-specific or global protein synthesis. Instead, eIF5A2 knockout leads to decreased intrinsic antiviral gene expression, including members of the IFITM and APOBEC3 family. Furthermore, cells lacking eIF5A2 display increased permissiveness to virus infection. Our results uncover eIF5A2 as a factor involved regulating the antiviral transcriptome, and reveal an example of how gene duplications of translation factors can result in proteins with distinct functions.

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

A phosphorylation-regulated eIF3d translation switch mediates cellular adaptation to metabolic stress.

Shutoff of global protein synthesis is a conserved response to cellular stresses. This general phenomenon is accompanied by the induction of distinct gene programs tailored to each stress. Although the mechanisms driving repression of general protein synthesis are well characterized, how cells reprogram the translation machinery for selective gene expression remains poorly understood. Here, we found that the noncanonical 5' cap-binding protein eIF3d was activated in response to metabolic stress in human cells. Activation required reduced CK2-mediated phosphorylation near the eIF3d cap-binding pocket. eIF3d controls a gene program enriched in factors important for glucose homeostasis, including members of the mammalian target of rapamycin (mTOR) pathway. eIF3d-directed translation adaptation was essential for cell survival during chronic glucose deprivation. Thus, this mechanism of translation reprogramming regulates the cellular response to metabolic stress.

CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.

A molecular filter for the cnidarian stinging response.

All animals detect and integrate diverse environmental signals to mediate behavior. Cnidarians, including jellyfish and sea anemones, both detect and capture prey using stinging cells called nematocytes which fire a venom-covered barb via an unknown triggering mechanism. Here, we show that nematocytes from Nematostella vectensis use a specialized voltage-gated calcium channel (nCaV) to distinguish salient sensory cues and control the explosive discharge response. Adaptations in nCaV confer unusually sensitive, voltage-dependent inactivation to inhibit responses to non-prey signals, such as mechanical water turbulence. Prey-derived chemosensory signals are synaptically transmitted to acutely relieve nCaV inactivation, enabling mechanosensitive-triggered predatory attack. These findings reveal a molecular basis for the cnidarian stinging response and highlight general principles by which single proteins integrate diverse signals to elicit discrete animal behaviors.

Repression of ferritin light chain translation by human eIF3.

A central problem in human biology remains the discovery of causal molecular links between mutations identified in genome-wide association studies (GWAS) and their corresponding disease traits. This challenge is magnified for variants residing in non-coding regions of the genome. Single-nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'-UTR) of the ferritin light chain (FTL) gene that cause hyperferritinemia are reported to disrupt translation repression by altering iron regulatory protein (IRP) interactions with the FTL mRNA 5'-UTR. Here, we show that human eukaryotic translation initiation factor 3 (eIF3) acts as a distinct repressor of FTL mRNA translation, and eIF3-mediated FTL repression is disrupted by a subset of SNPs in FTL that cause hyperferritinemia. These results identify a direct role for eIF3-mediated translational control in a specific human disease.

A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents.

Type II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational 'landscape' of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2ß) is additionally revealed to be significantly regulated by the enzymes' adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.

Modular Architecture of the STING C-Terminal Tail Allows Interferon and NF-κB Signaling Adaptation.

Stimulator of interferon genes (STING) is a key regulator of type I interferon and pro-inflammatory responses during infection, cellular stress, and cancer. Here, we reveal a mechanism for how STING balances activation of IRF3- and NF-κB-dependent transcription and discover that acquisition of discrete signaling modules in the vertebrate STING C-terminal tail (CTT) shapes downstream immunity. As a defining example, we identify a motif appended to the CTT of zebrafish STING that inverts the typical vertebrate signaling response and results in dramatic NF-κB activation and weak IRF3-interferon signaling. We determine a co-crystal structure that explains how this CTT sequence recruits TRAF6 as a new binding partner and demonstrate that the minimal motif is sufficient to reprogram human STING and immune activation in macrophage cells. Together, our results define the STING CTT as a linear signaling hub that can acquire modular motifs to readily adapt downstream immunity.

Bacterial cGAS-like enzymes synthesize diverse nucleotide signals.

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2',3' cGAMP Signaling.

In humans, the cGAS-STING immunity pathway signals in response to cytosolic DNA via 2',3' cGAMP, a cyclic dinucleotide (CDN) second messenger containing mixed 2'-5' and 3'-5' phosphodiester bonds. Prokaryotes also produce CDNs, but these are exclusively 3' linked, and thus the evolutionary origins of human 2',3' cGAMP signaling are unknown. Here we illuminate the ancient origins of human cGAMP signaling by discovery of a functional cGAS-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. Anemone cGAS appears to produce a 3',3' CDN that anemone STING recognizes through nucleobase-specific contacts not observed in human STING. Nevertheless, anemone STING binds mixed-linkage 2',3' cGAMP indistinguishably from human STING, trapping a unique structural conformation not induced by 3',3' CDNs. These results reveal that human mixed-linkage cGAMP achieves universal signaling by exploiting a deeply conserved STING conformational intermediate, providing critical insight for therapeutic targeting of the STING pathway.

eIF3 targets cell-proliferation messenger RNAs for translational activation or repression.

Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis and stress responses. The 13-subunit, 800-kilodalton eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). eIF3 binds to a highly specific program of messenger RNAs involved in cell growth control processes, including cell cycling, differentiation and apoptosis, via the mRNA 5' untranslated region. Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding the cell proliferation regulators c-JUN and BTG1 reveals that eIF3 uses different modes of RNA stem-loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis.

Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.

Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

Peroxisomes are signaling platforms for antiviral innate immunity.

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.