[Cytokine modification by the RNA regulatory protein TFL].

We found a novel gene, named "transformed follicular lymphoma" (TFL), in a patient who developed diffuse large B-cell lymphoma from follicular lymphoma. TFL modulates several cytokines and chemokines by binding their 3'UTR region of mRNA with its unique RNase motif. TFL is part of a family called zinc finger CCCH-type containing 12A-D. Due to its unique RNase motif, the TFL family is also called regulatory RNase (Regnase 1-4). TFL is expressed in lymphoid tissue and is upregulated by an inflammatory response, contributing to autoimmune diseases such as multiple sclerosis via IL-17 in CNS and worsening inflammation (cachexia) due to lymphoma progression through CXCL-13. Loss of TFL expression is reported to be a valuable biomarker for various cancers, including lung adenocarcinoma, endometrial cancer, and possibly lymphoma. In addition to its mRNA modulation function, Regnase1 is known to have deubiquitinase activity for TRAF2, 3, and 6, attenuating JNK and NFκB activity, and TFL captures and transports naked nonvesicular extracellular mRNA of IL-1ß to the nucleus, enhancing the tumor-killing activity in NK cells. Based on its potential to modulate inflammation, TFL could be a future treatment target for autoimmune diseases and cancer.

The antimicrobial protein RNase 7 directly restricts herpes simplex virus infection of human keratinocytes.

Approximately 22% of moderately to severely affected atopic dermatitis (AD) patients have a history of eczema herpeticum, a disseminated rash primarily caused by herpes simplex virus type 1 (HSV-1). Reduced activity of antimicrobial peptides may contribute to the increased susceptibility of AD patients to HSV-1. We previously demonstrated that the antimicrobial protein RNase 7 limits HSV-1 infection of human keratinocytes by promoting self-DNA sensing. Here, we addressed whether RNase 7 has any effect on HSV-1 infection when infecting keratinocytes without exogenously added costimulatory DNA, and which step(s) of the infection cycle RNase 7 interferes with. We quantified viral gene expression by RT-qPCR and flow cytometry, viral genome replication by qPCR, virucidal effects by plaque titration, and plaque formation and the subcellular localization of incoming HSV-1 particles by microscopy. Recombinant RNase 7 restricted HSV-1 gene expression, genome replication, and plaque formation in human keratinocytes. It decreased HSV-1 immediate-early transcripts independently of the induction of interferon-stimulated genes. Its main effect was on intracellular infection processes and not on extracellular virions or virus binding to cells. RNase 7 reduced the amount of cell-associated capsids and the HSV-1 envelope glycoprotein D at 3 but not at 0.5 h postinfection. Our data show that RNase 7 directly restricts HSV-1 infection of human keratinocytes, possibly by promoting the degradation of incoming HSV-1 particles. This suggests that RNase 7 may limit HSV-1 spread in the skin and that mechanisms that reduce its activity in the lesional skin of AD patients may increase their susceptibility to eczema herpeticum.

Conditional ablation of DIS3L2 ribonuclease in pre-meiotic germ cells causes defective spermatogenesis and infertility in male mice.

Rationale: Spermatogenesis is a highly organized cell differentiation process in mammals, involving mitosis, meiosis, and spermiogenesis. DIS3L2, which is primarily expressed in the cytoplasm, is an RNA exosome-independent ribonuclease. In female mice, Dis3l2-deficient oocytes fail to resume meiosis, resulting in arrest at the germinal vesicle stage and complete infertility. However, the role of DIS3L2 in germ cell development in males has remained largely unexplored. Methods: We established a pre-meiotic germ cell conditional knockout mouse model and investigated the biological function of DIS3L2 in spermatogenesis and male fertility through bulk RNA-seq and scRNA-seq analyses. Results: This study unveils that conditional ablation of Dis3l2 in pre-meiotic germ cells with Stra8-Cre mice impairs spermatogonial differentiation and hinders spermatocyte meiotic progression coupled with cell apoptosis. Such conditional ablation leads to defective spermatogenesis and sterility in adults. Bulk RNA-seq analysis revealed that Dis3l2 deficiency significantly disrupted the transcriptional expression pattern of genes related to the cell cycle, spermatogonial differentiation, and meiosis in Dis3l2 conditional knockout testes. Additionally, scRNA-seq analysis indicated that absence of DIS3L2 in pre-meiotic germ cells causes disrupted RNA metabolism, downregulated expression of cell cycle genes, and aberrant expression of spermatogonial differentiation genes, impeding spermatogonial differentiation. In meiotic spermatocytes, loss of DIS3L2 results in disturbed RNA metabolism, abnormal translation, and disrupted meiotic genes that perturb meiotic progression and induce cell apoptosis, leading to subsequent failure of spermatogenesis and male infertility. Conclusions: Collectively, these findings highlight the critical role of DIS3L2 ribonuclease-mediated RNA degradation in safeguarding the correct transcriptome during spermatogonial differentiation and spermatocyte meiotic progression, thus ensuring normal spermatogenesis and male fertility.

MCPIP1 promotes atrial remodeling by exacerbating miR-26a-5p/FRAT/Wnt axis-mediated atrial fibrosis in a rat model susceptible to atrial fibrillation.

Atrial fibrosis plays a critical role in the pathogenesis of atrial fibrillation (AF). Monocyte chemotactic protein-induced protein-1 (MCPIP1), recognized as a functional ribonuclease (RNase), exacerbates cardiac remodeling and contributes to a range of cardiovascular diseases. However, the involvement of MCPIP1 in atrial fibrosis and development of AF, along with its underlying biological mechanisms, remains poorly understood. This study demonstrated that knockdown of MCPIP1 significantly reduced AF inducibility, decreased left atrial diameter, and ameliorated atrial fibrosis, coinciding with reduced FRAT1/2/Wnt/ß-catenin signaling. Furthermore, the MCPIP1-D141N mutation attenuated AF vulnerability and atrial remodeling compared to MCPIP1 overexpression in an acetylcholine and calcium chloride (ACh-CaCl2)-induced rat model of AF. Conversely, overexpression of FRAT1/2 partially reversed the cardioprotective effects of MCPIP1-D141N mutation. Using H9C2 cell lines, we observed that MCPIP1 may induce a transcriptional effect that downregulates miR-26a-5p expression, and luciferase and RNA immunoprecipitation (RIP) assays substantiated the direct interaction between miR-26a-5p and FRAT1/2. Moreover, overexpression of miR-26a-5p countered MCPIP1-induced atrial remodeling and attenuated the progression of AF. In conclusion, these findings indicate that MCPIP1 facilitates atrial remodeling and the progression of AF by exacerbating miR-26a-5p/FRAT/Wnt axis-mediated atrial fibrosis through its RNase activity in an ACh-CaCl2-induced rat model of AF.

Discovery of Substituted 5-(2-Hydroxybenzoyl)-2-Pyridone Analogues as Inhibitors of the Human Caf1/CNOT7 Ribonuclease.

The Caf1/CNOT7 nuclease is a catalytic component of the Ccr4-Not deadenylase complex, which is a key regulator of post-transcriptional gene regulation. In addition to providing catalytic activity, Caf1/CNOT7 and its paralogue Caf1/CNOT8 also contribute a structural function by mediating interactions between the large, non-catalytic subunit CNOT1, which forms the backbone of the Ccr4-Not complex and the second nuclease subunit Ccr4 (CNOT6/CNOT6L). To facilitate investigations into the role of Caf1/CNOT7 in gene regulation, we aimed to discover and develop non-nucleoside inhibitors of the enzyme. Here, we disclose that the tri-substituted 2-pyridone compound 5-(5-bromo-2-hydroxy-benzoyl)-1-(4-chloro-2-methoxy-5-methyl-phenyl)-2-oxo-pyridine-3-carbonitrile is an inhibitor of the Caf1/CNOT7 nuclease. Using a fluorescence-based nuclease assay, the activity of 16 structural analogues was determined, which predominantly explored substituents on the 1-phenyl group. While no compound with higher potency was identified among this set of structural analogues, the lowest potency was observed with the analogue lacking substituents on the 1-phenyl group. This indicates that substituents on the 1-phenyl group contribute significantly to binding. To identify possible binding modes of the inhibitors, molecular docking was carried out. This analysis suggested that the binding modes of the five most potent inhibitors may display similar conformations upon binding active site residues. Possible interactions include π-π interactions with His225, hydrogen bonding with the backbone of Phe43 and Van der Waals interactions with His225, Leu209, Leu112 and Leu115.

Introducing synthetic thermostable RNase inhibitors to single-cell RNA-seq.

Single-cell RNA-sequencing (scRNAseq) is revolutionizing biomedicine, propelled by advances in methodology, ease of use, and cost reduction of library preparation. Over the past decade, there have been remarkable technical improvements in most aspects of single-cell transcriptomics. Yet, little to no progress has been made in advancing RNase inhibition despite maintained RNA integrity being critical during cell collection, storage, and cDNA library generation. Here, we demonstrate that a synthetic thermostable RNase inhibitor (SEQURNA) yields single-cell libraries of equal or superior quality compared to ubiquitously used protein-based recombinant RNase inhibitors (RRIs). Importantly, the synthetic RNase inhibitor provides additional unique improvements in reproducibility and throughput, enables new experimental workflows including retained RNase inhibition throughout heat cycles, and can reduce the need for dry-ice transports. In summary, replacing RRIs represents a substantial advancement in the field of single-cell transcriptomics.

HIV-1 controllers exhibit an enhanced antiretroviral innate state characterised by overexpression of p21 and MCPIP1 and silencing of ERVK-6 RNA expression.

BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

A ribonuclease T2 protein FocRnt2 contributes to the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA in vitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.

Genome-wide analysis of the NYN domain gene family in Brassica napus and its function role in plant growth and development.

The NYN domain gene family consists of genes that encode ribonucleases that are characterized by a newly identified NYN domain. Members of the family were widely distributed in all life kingdoms and play a crucial role in various RNA regulation processes, although the wide genome overview of the NYN domain gene family is not yet available in any species. Rapeseed (Brassica napus L.), a polyploid model species, is an important oilseed crop. Here, the phylogenetic analysis of these BnaNYNs revealed five distinct groups strongly supported by gene structure, conserved domains, and conserved motifs. The survey of the expansion of the gene family showed that the birth of BnaNYNs is explained by various duplication events. Furthermore, tissue-specific expression analysis, protein-protein interaction prediction, and cis-element prediction suggested a role for BnaNYNs in plant growth and development. Interestingly, the data showed that three tandem duplicated BnaNYNs (TDBs) exhibited distinct expression patterns from those other BnaNYNs and had a high similarity in protein sequence level. Furthermore, the analysis of one of these TDBs, BnaNYN57, showed that overexpression of BnaNYN57 in Arabidopsis thaliana and B. napus accelerated plant growth and significantly increased silique length, while RNA interference resulted in the opposite growth pattern. It suggesting a key role for the TDBs in processes related to plant growth and development.

RNase-mediated reprogramming of Yersinia virulence.

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.

Visual Evidence for the Recruitment of Four Enzymes with RNase Activity to the Bacillus subtilis Replication Forks.

Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5'->3' exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal.

Structural insights into RNA cleavage by a novel family of bacterial RNases.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.

Exploring the role of RNASET2 in the immune response of black soldier fly larvae.

T2 RNases are transferase-type enzymes distributed across phyla, crucial for breaking down single-stranded RNA molecules. In addition to their canonical function, several T2 enzymes exhibit pleiotropic roles, contributing to various biological processes, such as the immune response in invertebrates and vertebrates. This study aims at characterizing RNASET2 in the larvae of black soldier fly (BSF), Hermetia illucens, which are used for organic waste reduction and the production of valuable insect biomolecules for feed formulation and other applications. Given the exposure of BSF larvae to pathogens present in the feeding substrate, it is likely that the mechanisms of their immune response have undergone significant evolution and increased complexity. After in silico characterization of HiRNASET2, demonstrating the high conservation of this T2 homolog, we investigated the expression pattern of the enzyme in the fat body and hemocytes, two districts mainly involved in the insect immune response, in larvae challenged with bacterial infection. While no variation in HiRNASET2 expression was observed in the fat body following infection, a significant upregulation of HiRNASET2 synthesis occurred in hemocytes shortly after the injection of bacteria in the larva. The intracellular localization of HiRNASET2 in lysosomes of plasmatocytes, its extracellular association with bacteria, and the presence of a putative antimicrobial domain in the molecule, suggest its potential role in RNA clean-up and as an alarm molecule promoting phagocytosis activation by hemocytes. These insights contribute to the characterization of the immune response of Hermetia illucens larvae and may facilitate the development of animal feedstuff enriched with highly valuable BSF bioactive compounds.

Myo-inositol oxygenase CgMIOX3 alleviates S-RNase-induced inhibition of incompatible pollen tubes in pummelo.

Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype-specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes, and its downregulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid (AsA) accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity.

Keratinocyte and myeloid MCPIP1 have distinct roles in maintaining skin homeostasis.

The skin is a complex organ, and the intricate network between keratinocytes and immune cells is critical for ensuring skin function. Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) is a ribonuclease that functions as a key negative modulator of inflammation. We previously reported that conditional deletion of MCPIP1 in keratinocytes (Mcpip1EKO) impairs skin integrity in adult mice. A similar phenotype was observed following the depletion of MCPIP1 in the myeloid compartment (Mcpip1MKO). The aim of this study was to develop a keratinocyte and myeloid double-MCPIP1 knockout mouse model to clarify the specific roles of myeloid and epidermal MCPIP1 in skin biology. Histological analyses indicated that the skin morphology changed after depletion of MCPIP1 in cells of myeloid origin as well as in keratinocytes. The thicknesses of the epidermal and subcutaneous fat layers increased in the mice with a loss of epidermal MCPIP1, whereas the loss of myeloid MCPIP1 had the opposite effect. In addition, both types of mice showed opposite responses to stimulation with 12-O-tetradecanoylphorbol-13-acetate. Transcriptomic profiling of whole-skin lysates revealed some common target transcripts in all the knockout mice. Further analyses revealed that distinct pathways are modulated following the loss of epidermal or myeloid MCPIP1. The skin morphology and inflammatory phenotype of keratinocyte and myeloid double-MCPIP1 knockout mice resembled those of mice with only keratinocyte-specific knockout of MCPIP1. Overall, myeloid and epidermal MCPIP1 play important but distinct roles in the modulation of skin-related processes.

A comparative analysis of mycobacterial ribonucleases: Towards a therapeutic novel drug target.

Bacterial responses to continuously changing environments are addressed through modulation of gene expression at the level of transcription initiation, RNA processing and/or decay. Ribonucleases (RNases) are hydrolytic or phosphorolytic enzymes involved in a majority of RNA metabolism reactions. RNases play a crucial role in RNA degradation, either independently or in collaboration with various trans-acting regulatory factors. The genus Mycobacterium consists of five subgenera: Mycobacteroides, Mycolicibacterium, Mycobacterium, Mycolicibacter and Mycolicibacillus, which include 63 fully sequenced species (pathogenic/non-pathogenic) to date. These include 13 different RNases, among which 5 are exonucleases (RNase PH, PNPase, RNase D, nano-RNases and RNase AS) and 8 are endonucleases (RNase J, RNase H, RNase P, RNase III, RNase BN, RNase Z, RNase G and RNase E), although RNase J and RNase BN were later identified to have exoribonuclease functions also. Here, we provide a detailed comparative insight into the Escherichia coli and mycobacterial RNases with respect to their types, phylogeny, structure, function, regulation and mechanism of action, with the main emphasis on RNase E. Among these 13 different mycobacterial RNases, 10 are essential for cell survival and have diverse structures hence, they are promising drug targets. RNase E is also an essential endonuclease that is abundant in many bacteria, forms an RNA degradosome complex that controls central RNA processing/degradation and has a conserved 5' sensor domain/DNase-I like region in its RNase domain. The essential mycobacterial RNases especially RNase E provide a potential repertoire of drug targets that can be exploited for inhibitor/modulator screening against many deadly mycobacterial diseases.

Colletotrichum fructicola co-opts cytotoxic ribonucleases that antagonize host competitive microorganisms to promote infection.

Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus Colletotrichum fructicola, which causes pear anthracnose, can colonize different plant tissues like leaves and fruits, which are occupied by a diversity of microbes. We speculate that this fungus produces antimicrobial effectors to outcompete host-associated competitive microorganisms. Herein, we identified two secreted ribonucleases, CfRibo1 and CfRibo2, from the C. fructicola secretome. The two ribonucleases both possess ribonuclease activity and showed cytotoxicity in Nicotianan benthamiana without triggering immunity in an enzymatic activity-dependent manner. CfRibo1 and CfRibo2 recombinant proteins exhibited toxicity against Escherichia coli, Saccharomyces cerevisiae, and, importantly, the phyllosphere microorganisms isolated from the pear host. Among these isolated microbial strains, Bacillus altitudinis is a pathogenic bacterium causing pear soft rot. Strikingly, CfRibo1 and CfRibo2 were found to directly antagonize B. altitudinis to facilitate C. fructicola infection. More importantly, CfRibo1 and CfRibo2 functioned as essential virulence factors of C. fructicola in the presence of host-associated microorganisms. Further analysis revealed these two ribonucleases are widely distributed in fungi and are undergoing purifying selection. Our results provide the first evidence of antimicrobial effectors in Colletotrichum fungi and extend the functional diversity of fungal ribonucleases in plant-pest-environment interactions. IMPORTANCE: Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in various crops in agriculture, and understanding how this fungus establishes successful infection is of great significance for anthracnose disease management. Fungi are known to produce secreted effectors as weapons to promote virulence. Considerable progress has been made in elucidating how effectors manipulate plant immunity; however, their importance in modulating environmental microbes is frequently neglected. The present study identified two secreted ribonucleases, CfRibo1 and CfRibo2, as antimicrobial effectors of C. fructicola. These two proteins both possess toxicity to pear phyllosphere microorganisms, and they efficiently antagonize competitive microbes to facilitate the infection of pear hosts. This study represents the first evidence of antimicrobial effectors in Colletotrichum fungi, and we consider that CfRibo1 and CfRibo2 could be targeted for anthracnose disease management in diverse crops in the future.

Ccr4-not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy.

The Ccr4-Not complex contains the poorly understood Not4 ubiquitin ligase that functions in transcription, mRNA decay, translation, proteostasis, and endolysosomal nutrient signaling. To gain further insight into the in vivo functions of the ligase, we performed quantitative proteomics in Saccharomyces cerevisiae using yeast cells lacking Not4, or cells overexpressing wild-type Not4 or an inactive Not4 mutant. Herein, we provide evidence that balanced Not4 activity maintains ribosomal protein (RP) homeostasis independent of changes to RP mRNA or known Not4 ribosomal substrates. Intriguingly, we also find that Not4 loss activates 40S ribosomal autophagy independently of canonical Atg7-dependent macroautophagy, indicating that microautophagy is responsible. We previously demonstrated that Ccr4-Not stimulates the target of rapamycin complex 1 (TORC1) signaling, which activates RP expression and inhibits autophagy, by maintaining vacuole V-ATPase H+ pump activity. Importantly, combining Not4 deficient cells with a mutant that blocks vacuole H+ export fully restores RP expression and increases 40S RP autophagy efficiency. In contrast, restoring TORC1 activity alone fails to rescue either process, indicating that Not4 loss disrupts additional endolysosomal functions that regulate RP expression and 40S autophagy. Analysis of the Not4-regulated proteome reveals increases in endolysosomal and autophagy-related factors that functionally interact with Not4 to control RP expression and affect 40S autophagy. Collectively, our data indicate that balanced Ccr4-Not ubiquitin ligase signaling maintains RP homeostasis and inhibits 40S autophagy via the ligase's emerging role as an endolysosomal regulator.

Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system.

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.

Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.