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Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.

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Discovery of CARF (Collaborator of ARF)/CDKN2AIP as an ARF-interacting protein that promotes ARF-p53-p21WAF1 signaling and cellular senescence, initially established its role in genomic stress. Multiple reports further unraveled its role in regulation of senescence, growth arrest, apoptosis, or malignant transformation of cells in response to a variety of stress conditions in cultured human cells. It has been established as an essential protein. Whereas CARF-compromised cells undergo apoptosis, its enrichment has been recorded in a variety of cancer cells and has been associated with malignant transformation. We earlier demonstrated its role in stress-induced cell phenotypes that ranged from growth arrest, apoptosis, or malignant transformation. In the present study, we assessed the molecular mechanism of quantitative impact of change in CARF expression level on these cell fates. Stress-induced changes in CARF expression were assessed quantitatively with proteins involved in proteotoxicity, oxidative, genotoxic, and cytotoxic stress. These comparative quantitative analyses confirmed that (i) CARF responds to diverse stresses in a quantitative manner, (ii) its expression level serves as a reliable predictive measure of cell fates (iii) it correlates more with the DNA damage and MDA levels than the oxidative and proteotoxic signatures and (iv) CARF-expression based quantitative assay may be recruited for stress diagnostic applications.

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CARF (CDKN2AIP) regulates cellular fate in response to various stresses. However, its role in metabolic stress is unknown. We found that fatty livers from mice exhibit low CARF expression. Similarly, overloaded palmitate inhibited CARF expression in HepG2 cells, suggesting that excess fat-induced stress downregulates hepatic CARF. In agreement with this, silencing and overexpressing CARF resulted in higher and lower fat accumulation in HepG2 cells, respectively. Furthermore, CARF overexpression lowered the ectopic palmitate accumulation in HepG2 cells. We were interested in understanding the role of hepatic CARF and underlying mechanisms in the development of NAFLD. Mechanistically, transcriptome analysis revealed that endoplasmic reticulum (ER) stress and oxidative stress pathway genes significantly altered in the absence of CARF. IRE1α, GRP78, and CHOP, markers of ER stress, were increased, and the treatment with TUDCA, an ER stress inhibitor, attenuated fat accumulation in CARF-deficient cells. Moreover, silencing CARF caused a reduction of GPX3 and TRXND3, leading to oxidative stress and apoptotic cell death. Intriguingly, CARF overexpression in HFD-fed mice significantly decreased hepatic steatosis. Furthermore, overexpression of CARF ameliorated the aberrant ER function and oxidative stress caused by fat accumulation. Our results further demonstrated that overexpression of CARF alleviates HFD-induced insulin resistance assessed with ITT and GTT assay. Altogether, we conclude that excess fat-induced reduction of CARF dysregulates ER functions and lipid metabolism leading to hepatic steatosis.

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Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

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Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles in membrane organization, fluidity, signaling, and antioxidative functions, and abnormal plasmalogen levels correlate with various human pathologies, including cancer and Alzheimer's disease. The presence of plasmalogens in animals and in anaerobic bacteria, but not in plants and fungi, is well-documented. However, their occurrence in the obligately aerobic myxobacteria, exceptional among aerobic bacteria, is often overlooked. Tellingly, discovery of the key desaturase indispensable for vinyl ether bond formation, and therefore fundamental in plasmalogen biogenesis, emerged from delving into how the soil myxobacterium Myxococcus xanthus responds to light. A recent pioneering study unmasked myxobacterial CarF and its human ortholog TMEM189 as the long-sought plasmanylethanolamine desaturase (PEDS1), thus opening a crucial door to study plasmalogen biogenesis, functions, and roles in disease. The findings demonstrated the broad evolutionary sweep of the enzyme and also firmly established a specific signaling role for plasmalogens in a photooxidative stress response. Here, we will recount our take on this fascinating story and its implications, and review the current state of knowledge on plasmalogens, their biosynthesis and functions in the aerobic myxobacteria.

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BACKGROUND: Artificial intelligence (AI) shows great potential to streamline the treatment planning process. However, its clinical adoption is slow due to the limited number of clinical evaluation studies and because often, the translation of the predicted dose distribution to a deliverable plan is lacking. This study evaluates two different, deliverable AI plans in terms of their clinical acceptability based on quantitative parameters and qualitative evaluation by four radiation oncologists. METHODS: For 20 left-sided node-negative breast cancer patients, treated with a prescribed dose of 40.05 Gy, using tangential beam intensity modulated radiotherapy, two model-based treatment plans were evaluated against the corresponding manual plan. The two models used were an in-house developed U-net model and a vendor-developed contextual atlas regression forest model (cARF). Radiation oncologists evaluated the clinical acceptability of each blinded plan and ranked plans according to preference. Furthermore, a comparison with the manual plan was made based on dose volume histogram parameters, clinical evaluation criteria and preparation time. RESULTS: The U-net model resulted in a higher average and maximum dose to the PTV (median difference 0.37 Gy and 0.47 Gy respectively) and a slightly higher mean heart dose (MHD) (0.01 Gy). The cARF model led to higher average and maximum doses to the PTV (0.30 and 0.39 Gy respectively) and a slightly higher MHD (0.02 Gy) and mean lung dose (MLD, 0.04 Gy). The maximum MHD/MLD difference was ≤ 0.5 Gy for both AI plans. Regardless of these dose differences, 90-95% of the AI plans were considered clinically acceptable versus 90% of the manual plans. Preferences varied between the radiation oncologists. Plan preparation time was comparable between the U-net model and the manual plan (287 s vs 253 s) while the cARF model took longer (471 s). When only considering user interaction, plan generation time was 121 s for the cARF model and 137 s for the U-net model. CONCLUSIONS: Two AI models were used to generate deliverable plans for breast cancer patients, in a time-efficient manner, requiring minimal user interaction. Although the AI plans resulted in slightly higher doses overall, radiation oncologists considered 90-95% of the AI plans clinically acceptable.

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RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

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Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating "ring nuclease" activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.

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Myxobacteria are Gram-negative δ-proteobacteria found predominantly in terrestrial habitats and often brightly colored due to the biosynthesis of carotenoids. Carotenoids are lipophilic isoprenoid pigments that protect cells from damage and death by quenching highly reactive and toxic oxidative species, like singlet oxygen, generated upon growth under light. The model myxobacterium Myxococcus xanthus turns from yellow in the dark to red upon exposure to light because of the photoinduction of carotenoid biosynthesis. How light is sensed and transduced to bring about regulated carotenogenesis in order to combat photooxidative stress has been extensively investigated in M. xanthus using genetic, biochemical and high-resolution structural methods. These studies have unearthed new paradigms in bacterial light sensing, signal transduction and gene regulation, and have led to the discovery of prototypical members of widely distributed protein families with novel functions. Major advances have been made over the last decade in elucidating the molecular mechanisms underlying the light-dependent signaling and regulation of the transcriptional response leading to carotenogenesis in M. xanthus. This review aims to provide an up-to-date overview of these findings and their significance.

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In prokaryotes, CRISPR-Cas immune systems recognise and cleave foreign nucleic acids to defend against Mobile Genetic Elements (MGEs). Type III CRISPR-Cas complexes also synthesise cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defence. In particular, cOA-stimulated nucleases degrade RNA and DNA non-specifically, which slows MGE replication but also impedes cell growth, necessitating mechanisms to eliminate cOA in order to mitigate collateral damage. Extant cOA is degraded by a new class of enzyme termed a 'ring nuclease', which cleaves cOA specifically and switches off CRISPR ancillary enzymes. Several ring nuclease families have been characterised to date, including a family used by MGEs to circumvent CRISPR immunity, and encompass diverse protein folds and distinct cOA cleavage mechanisms. In this review we outline cOA signalling, discuss how different ring nucleases regulate the cOA signalling pathway, and reflect on parallels between cyclic nucleotide-based immune systems to reveal new areas for exploration.

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BACKGROUND: Even though the relevance of microRNA (miR)-302c has been studied, little is known about its involvement in colon cancer (CC). AIMS: Our aim here was to investigate the role of miR-302c in the cancer stem cells (CSCs) of CC. METHODS: Firstly, the CSCs were screened out from cultured SW1116 and SW480 cells by flow cytometry, and the differentially expressed miRNAs in cell were obtained by microarray analysis. The expression of miR-302c, collaborator of ARF (CARF), and Wnt/ß-catenin-related genes in CSCs was determined by means of RT-qPCR and Western blot. A dual-luciferase reporter assay was conducted to authenticate the binding relationship between miR-302c and CARF. Proliferation, migration, invasion, sphere formation as well as apoptosis of CSCs were assessed by cell counting kit-8, Transwell assay, sphere formation assay as well as flow cytometric analysis, respectively. The roles of miR-302c and CARF in tumor growth were determined in vivo. RESULTS: The expression of miR-302c in CC cells was reduced versus that in normal cells. The overexpression of miR-302c weakened the stemness, proliferation, invasion, and migration abilities while induced apoptosis of CSCs in CC. Also, miR-302c reduced tumor size and weight in mice, accompanied with lowered CARF expression. The mechanistic analysis manifested that miR-302c bound to CARF and suppressed its expression and disrupted the Wnt/ß-catenin signaling. CONCLUSION: This study offers a novel characterization of miR-302c function in CSCs in CC, which may be beneficial to the development of capable therapeutic options for CC.

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Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.

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Cas10 is the signature gene for type III CRISPR-Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn2+-dependent deadenylation exoribonuclease. However, its specific role in CRISPR-Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR-Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

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Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

Bacteria protect themselves from infections using a system called CRISPR-Cas, which helps the cells to detect and destroy invading threats. The type III CRISPR-Cas system, in particular, is one of the most widespread and efficient at killing viruses. When a bacterium is infected, the CRISPR-Cas system takes a fragment of the genetic material of the virus, and copies it into a molecule. These molecular 'police mugshots' are then loaded into a complex of Cas proteins that patrol the cell, looking for a match and destroying any virus that can be identified. Some Cas proteins also produce alarm signals, called cyclic oligoadenylates (cOAs), which can trigger additional defences. However, this process can damage the genetic material of the bacterium, harming or even killing the cell. Enzymes known as ring nucleases can promptly degrade cOAs and turn off this defence system before it causes harm. However, ring nucleases have only been found in a few species to date; how most bacteria deal with cOA toxicity has remained unknown. Here, Athukoralage et al. set out to determine whether a widespread enzyme known as Csx3, which is often associated with type III CRISPR-Cas systems, could be an alternative off switch for cOA triggered defences. Initial 'test tube' experiments with purified Csx3 proteins confirmed that the enzyme could indeed break down cOAs. A careful dissection of Csx3's molecular structure, using biochemical and biophysical techniques, revealed that it worked by 'sandwiching' a cOA molecule between two co-operating portions of the enzyme. As a final test, Csx3 was introduced into strains of bacteria genetically engineered to have a fully functional Type III CRISPR-Cas system. In these cells, Csx3 successfully turned off the Type III immune response. These results reveal a new way that bacteria avoid the toxic side effects of their own immune defences. Ultimately, this could pave the way for the development of anti-bacterial drugs that work by blocking Csx3 or similar proteins.

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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.

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CARF (Collaborator of ARF) was discovered as an ARF-interacting protein that activated ARF-p53-p21WAF1 signaling involved in cellular response to a variety of stresses, including oxidative, genotoxic, oncogenic, or telomere deprotection stresses, leading to senescence, growth arrest, or apoptosis. Of note, whereas suppression of CARF was lethal, its enrichment was associated with increased proliferation and malignant transformation of cells. These reports have predicted that CARF could serve as a multi-stress marker with a predictive value for cell fates. Here, we recruited various in vitro stress models and examined their effect on CARF expression using human normal fibroblasts. We demonstrate that CARF levels in stress and post-stress conditions could predict the fate of cells towards either death or enhanced proliferation and malignant transformation. We provide extensive molecular evidence that (i) CARF expression changes in response to stress, (ii) it modulates cell death or survival signaling and determines the fate of cells, and (iii) it may serve as a predictive measure of cellular response to stress and an important marker for biosafety.

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Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently.

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INTRODUCTION: The role of CARF, a calcium-responsive transcription factor, in colorectal cancer initiation and development is still unknown. Here, we report that CARF promotes colorectal cancer stemness through ERBB signaling pathway. MATERIALS AND METHODS: Both colorectal cancer cell lines and primary cells were used in this study. The levels of target mRNA and protein in the cells were examined by qRT-PCR and Western blot. Gene manipulation was achieved by the lentivirus delivery system. Luciferase reporter gene assay was employed to analyze the transcriptional activity of the promoter. ChIP assay was performed for the examination of the binding between CARF and the promoters of MAPK8 and JUN. Kaplan-Meier survival curve was generated by the R2 program. Correlation analysis was performed using Spearman correlation analysis. RESULTS: Aberrant upregulation of CARF has been found in tumor tissues of colorectal cancer patients and associated with poor prognosis. Ectopic expression of CARF promoted the sphere-formation activities, as well as the expression of stem cell markers in colorectal cancer cells and knockdown of CARF, inhibited these activities. The mechanistic analysis showed that CARF directly binds to the promoter of MAPK8 and JUN, promotes the expression of MAPK8 and JUN, activates the ERBB signaling pathway, and thereby promotes the maintenance of the stemness in colorectal cancer cells. CONCLUSION: CARF, as an oncogene, promotes colorectal cancer stemness by activating ERBB signaling pathway. The ERBB signaling pathway that serves as the main downstream effector of CARF could be an efficient drug target for colorectal cancer caused by aberrant expression of CARF.

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Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.

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Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.

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