Ribonuclease Activity Research Articles

Abstract B033: Targeting acquired resistance to HIF2α inhibition in kidney cancer

Abstract Recently, a HIF-2α inhibitor MK-6482 was developed and approved by FDA to treat certain patients with VHL disease-associated RCC. However, in preclinical studies, both intrinsic and adaptive resistance to this drug was reported, which highlights the necessity of uncovering the mechanisms underlying drug resistance and developing alternative treatments for HIF-2α inhibitor-resistant patients. To this end, we established several 786-O cell lines that are resistant to PT2399 (analog of MK-6482) treatment. To characterize the differences between sensitive and resistant cell lines, we performed RNA sequencing, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and CUT&RUN sequencing experiments. Through integrative analyses of all sequencing data, we identified a series of candidate genes which might contribute to the acquirement of drug resistance for further validation. We identified 2'-5'-oligoadenylate synthetase 2 (OAS2) as one of the candidate genes exhibiting both significant upregulation in RNA level and dramatically increased chromatin accessibility in the promoter region in resistant lines comparing to sensitive lines. We further validated the upregulation of OAS2 mRNA level as well as protein level in all resistant clones by RT-qPCR and western blot, respectively. OAS2 belongs to 2-5A synthetase family, which is involved in the activation of latent RNase L and degradation of viral RNA during innate immune response to viral infection. Interestingly, the other 3 members in this protein family, OAS1, OAS3 and OASL, are also significantly upregulated at RNA level in resistant cell lines, although the upregulation of chromatin accessibility is modest. Furthermore, OAS2, along with the other 3 family members, all show increased expression levels in ccRCC tumor tissues comparing to normal tissues. Therefore, we hypothesize that OAS2 protein promotes ccRCC cell proliferation under HIF-2α inhibitor treatment, thus contributing to the adaptive resistance to HIF-2α inhibitor. We will test this hypothesis through a series of loss-of-function and gain-of-function experiments, while continuing to investigate other candidate genes. Citation Format: Fangzhou Zhao, Jeremy Simon, Qing Zhang. Targeting acquired resistance to HIF2α inhibition in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B033.

RNase E biomolecular condensates stimulate PNPase activity

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3′-5′ exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase’s activity is stimulated within the BR-body. We reconstituted RNase E’s C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase’s catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein–protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E’s unique biomolecular condensate environment tuned PNPase’s substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E’s biomolecular condensates.

Rege-1 promotes C. elegans survival by modulating IIS and TOR pathways.

Metabolic pathways are known to sense the environmental stimuli and result in physiological adjustments. The responding processes need to be tightly controlled. Here, we show that upon encountering P. aeruginosa, C. elegans upregulate the transcription factor ets-4, but this upregulation is attenuated by the ribonuclease, rege-1. As such, mutants with defective REGE-1 ribonuclease activity undergo ets-4-dependent early death upon challenge with P. aeruginosa. Furthermore, mRNA-seq analysis revealed associated global changes in two key metabolic pathways, the IIS (insulin/IGF signaling) and TOR (target of rapamycin) kinase signaling pathways. In particular, failure to degrade ets-4 mRNA in activity-defective rege-1 mutants resulted in upregulation of class II longevity genes, which are suppressed during longevity, and activation of TORC1 kinase signaling pathway. Genetic inhibition of either pathway way was sufficient to abolish the poor survival phenotype in rege-1 worms. Further analysis of ETS-4 ChIP data from ENCODE and characterization of one upregulated class II gene, ins-7, support that the Class II genes are activated by ETS-4. Interestingly, deleting an upregulated Class II gene, acox-1.5, a peroxisome β-oxidation enzyme, largely rescues the fat lost phenotype and survival difference between rege-1 mutants and wild-types. Thus, rege-1 appears to be crucial for animal survival due to its tight regulation of physiological responses to environmental stimuli. This function is reminiscent of its mammalian ortholog, Regnase-1, which modulates the intestinal mTORC1 signaling pathway.

Mapping the MOB proteins’ proximity network reveals a unique interaction between human MOB3C and the RNase P complex

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

Analysis of ribonuclease and peroxidase activities during maize ( Zea mays) response to Meloidogyne arenaria infection

Analysis of ribonuclease and peroxidase activities during maize ( Zea mays) response to Meloidogyne arenaria infection

Inhibition of IRE1 RNase activity modulates tumor cell progression and enhances the response to chemotherapy in colorectal cancer.

Drug resistance is one of the clinical challenges that limits the effectiveness of chemotherapy. Recent reports suggest that the unfolded protein response (UPR) and endoplasmic reticulum stress-adaptation signalling pathway, along with increased activation of its inositol-requiring enzyme 1α (IRE1α) arm, may be contributors to the pathogenesis of colorectal cancer (CRC). Here, we aimed to target the IRE1α/XBP1 pathway in order to sensitise CRC cells to the effects of chemotherapy.The CT26 colorectal cell line was treated with tunicamycin, and then was exposed to different concentrations of 5-fluorouracil (5-FU), either alone and/or in combination with the IRE1α inhibitor, 4µ8C. An MTT assay, flow cytometry and RT-PCR were performed to determine cell growth, apoptosis and IRE1α activity, respectively. In vivo BALB/c syngeneic colorectal mice received chemotherapeutic drugs. Treatment responses, tumour sizes and cytotoxicity were assessed via a range of pathological tests.4µ8C was found to inhibit the growth of CRC, at a concentration of 10µg/ml, without detectable cytotoxic effects and also significantly enhanced the cytotoxic potential of 5-FU, in CRC cells. In vivo experiments revealed that 4µ8C, at a concentration of 50µM/kg prevented tumour growth without any cytotoxic or metastatic effects. Interestingly, the combination of 4µ8C with 5-FU remarkably enhanced drug responses, up to 40-60% and also lead to significantly greater inhibition of tumour growth, in comparison to monotherapy, in CRC mice.Targeting the IRE1α/XBP1 axis of the UPR could enhance the effectiveness of chemotherapy in both in vitro and in vivo models of CRC.

Micro RNAs: The Game Changers of Diagnosis and Therapy of Human Cardiovascular Diseases

MicroRNAs (miRNA) are 21-23 nucleotide-long non-protein-coding RNAs discovered in C. elegans in 1993 that resist endogenous RNase activity. More than one hundred miRNAs were discovered in samples from healthy humans and identified as circulating miRNAs. These small RNAs function as regulatory elements in many cellular events. miRNAs act by reducing the expression of the target gene.Although the use of miRNAs in cardiovascular health has not yet been supported by clinical trials, recent studies have suggested that they can be used as clinical biomarkers in the diagnosis and treatment of cardiovascular diseases. However, the promising results of current studies support potential future applications of miRNA therapeutics. In this paper, the action mechanisms of miRNAs and their potential use as a biomarker in the diagnosis and treatment of cardiovascular diseases are mentioned.

The ribonuclease domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

The SLFN11 gene participates in cell fate decision following cancer chemotherapy and encodes the N-terminal ribonuclease (RNase) domain and the C-terminal helicase/ATPase domain. How these domains contribute to the chemotherapeutic response remains controversial. Here, we expressed SLFN11 containing mutations in two critical residues required for RNase activity in SLFN11-/- cells. We found that this mutant was still able to suppress DNA damage tolerance, destabilized the stalled replication forks, and perturbed recruitment of the fork protector RAD51. In contrast, we confirmed that the helicase domain was essential to accelerate fork degradation. The fork degradation by the RNase mutant was dependent on both DNA2 and MRE11 nuclease, but not on MRE11's novel interactor FXR1. Collectively, these results supported the view that the RNase domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

Adenosine A2A Receptor Regulates microRNA-181b Expression in Aorta: Therapeutic Implications for Large-Artery Stiffness.

Background The identification of large-artery stiffness as a major, independent risk factor for cardiovascular disease-associated morbidity and death has focused attention on identifying therapeutic strategies to combat this disorder. Genetic manipulations that delete or inactivate the translin/trax microRNA-degrading enzyme confer protection against aortic stiffness induced by chronic ingestion of high-salt water (4%NaCl in drinking water for 3 weeks) or associated with aging. Therefore, there is heightened interest in identifying interventions capable of inhibiting translin/trax RNase activity, as these may have therapeutic efficacy in large-artery stiffness. Methods and Results Activation of neuronal adenosine A2A receptors (A2ARs) triggers dissociation of trax from its C-terminus. As A2ARs are expressed by vascular smooth muscle cells (VSMCs), we investigated whether stimulation of A2AR on vascular smooth muscle cells promotes the association of translin with trax and, thereby increases translin/trax complex activity. We found that treatment of A7r5 cells with the A2AR agonist CGS21680 leads to increased association of trax with translin. Furthermore, this treatment decreases levels of pre-microRNA-181b, a target of translin/trax, and those of its downstream product, mature microRNA-181b. To check whether A2AR activation might contribute to high-salt water-induced aortic stiffening, we assessed the impact of daily treatment with the selective A2AR antagonist SCH58261 in this paradigm. We found that this treatment blocked aortic stiffening induced by high-salt water. Further, we confirmed that the age-associated decline in aortic pre-microRNA-181b/microRNA-181b levels observed in mice also occurs in humans. Conclusions These findings suggest that further studies are warranted to evaluate whether blockade of A2ARs may have therapeutic potential in treating large-artery stiffness.

Mechanisms of Regulation of Cryptic Prophage-Encoded Gene Products in Escherichia coli.

The dicBF operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. In this study, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. IMPORTANCE Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.

Mechanistic studies on substrate inhibition and substrate activation of ribonuclease A: experimental and in silico investigation

Ribonuclease A (RNase A) is an endonuclease that plays a significant role in antimicrobial activity by the cleavage and hydrolysis of ssRNA. In this study, the interactions between RNase A and cCMP have been investigated, through molecular docking and a 200 ns molecular dynamics simulation. The enzyme kinetic properties were analyzed using saturation curve, Eadie-Hofstee, and Hill plots. The docking results indicate that the cCMP-RNase A complexes are stabilized through hydrophobic interaction, hydrogen bonding, and π-π stacking interaction. The most binding site is observed in the catalytic cleft, especially at residue His12 and His119. Enzyme-ligand docking study indicates that cCMP binds to residues located in the internal cavity of the catalytic site and shows three phases of binding modes. The analysis of MD simulations shows that RMSD, Rg, and RMSF reach equilibrium. The ΔGbinding of the cCMP-RNase A was negative (-619.673 kJ/mol), The comparison between the results pre and post MD simulation showed that ΔGbinding after MD simulation causes to more stable the structure than before simulation. Experimental methods such as saturation, Eadie-Hofstee, and Hill plots confirm theoretical data and show three phases of binding modes as well. Two significant events are demonstrated in the interaction between RNase A and cCMP: substrate activation and substrate inhibition are observed in concentrations below and above 0.8 mM, respectively, for cCMP. Choosing the appropriate concentration of cCMP is very important in investigation of ribonuclease A’s catalytic behavour, especially for exploration of the effects of some drugs on biological behaviours related to ribonuclease A. Communicated by Ramaswamy H. Sarma

Bacillus licheniformis: A Producer of Antimicrobial Substances, including Antimycobacterials, Which Are Feasible for Medical Applications.

Bacillus licheniformis produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of B. licheniformis to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria.

A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins

Organellar C-to-U RNA editing in plants occurs in complexes composed of various classes of nuclear-encoded proteins. DYW-deaminases are zinc metalloenzymes that catalyze hydrolytic deamination required for C-to-U modification editing. Solved crystal structures for DYW-deaminase domains display all structural features consistent with a canonical cytidine deamination mechanism. However, some recombinant DYW-deaminases from plants have been associated with ribonuclease activity in vitro. Direct ribonuclease activity by an editing factor is confounding since it is not required for deamination of cytosine, theoretically would be inimical for mRNA editing, and does not have a clear physiological function in vivo. His-tagged recombinant DYW1 from Arabidopsis thaliana (rAtDYW1) was expressed and purified using immobilized metal affinity chromatography (IMAC). Fluorescently labeled RNA oligonucleotides were incubated with recombinant AtDYW1 under different conditions. Percent relative cleavage of RNA probes was recorded at multiple time points from triplicate reactions. The effects of treatment with zinc chelators EDTA and 1, 10-phenanthroline were examined for rAtDYW1. Recombinant His-tagged RNA editing factors AtRIP2, ZmRIP9, AtRIP9, AtOZ1, AtCRR4, and AtORRM1 were expressed in E. coli and purified. Ribonuclease activity was assayed for rAtDYW1 in the presence of different editing factors. Lastly, the effects on nuclease activity in the presence of nucleotides and modified nucleosides were investigated. RNA cleavage observed in this study was linked to the recombinant editing factor rAtDYW1 in vitro. The cleavage reaction is sensitive to high concentrations of zinc chelators, indicating a role for zinc ions for activity. The addition of equal molar concentrations of recombinant RIP/MORF proteins reduced cleavage activity associated with rAtDYW1. However, addition of equal molar concentrations of purified recombinant editing complex proteins AtCRR4, AtORRM1, and AtOZ1 did not strongly inhibit ribonuclease activity on RNAs lacking an AtCRR4 cis-element. Though AtCRR4 inhibited AtDYW1 activity for oligonucleotides with a cognate cis-element. The observation that editing factors limit ribonuclease activity of rAtDYW1 in vitro, suggests that nuclease activities are limited to RNAs in absence of native editing complex partners. Purified rAtDYW1 was associated with the hydrolysis of RNA in vitro, and activity was specifically inhibited by RNA editing factors.

Structural and Biophysical properties of therapeutically important proteins Rv1509 and Rv2231A of Mycobacterium tuberculosis

Through comparative analyses using BLASTp and BLASTn of the 25 target sequences, our research identified two unique post-transcriptional modifiers, Rv1509 and Rv2231A, which serve as distinctive and characteristic proteins of M.tb - the Signature Proteins. Here, we have characterized these two signature proteins associated with pathophysiology of M.tb which may prove to be therapeutically important targets. Dynamic Light Scattering and Analytical Gel Filtration Chromatography exhibited that Rv1509 exists as a monomer while Rv2231A as a dimer in solution. Secondary structures were determined using Circular Dichroism and further validated through Fourier Transform Infrared spectroscopy. Both the proteins are capable of withstanding a wide range of temperature and pH variations. Fluorescence spectroscopy based binding affinity experiments showed that Rv1509 binds to iron and may promote organism growth by chelating iron. In the case of Rv2231A, a high affinity for its substrate RNA was observed, which is facilitated in presence of Mg2+ suggesting it might have RNAse activity, supporting the prediction through in-silico studies. This is the first study on biophysical characterization of these two therapeutically important proteins, Rv1509 and Rv2231A, providing important insights into their structure -function correlations which are crucial for development of new drugs/ early diagnostics tools targeting these proteins.

The Unfolded Protein Response Sensor IRE1 Regulates Activation of In Vitro Differentiated Type 1 Conventional DCs with Viral Stimuli.

Type 1 conventional dendritic cells (cDC1s) are leukocytes competent to coordinate antiviral immunity, and thus, the intracellular mechanisms controlling cDC1 function are a matter of intense research. The unfolded protein response (UPR) sensor IRE1 and its associated transcription factor XBP1s control relevant functional aspects in cDC1s including antigen cross-presentation and survival. However, most studies connecting IRE1 and cDC1 function are undertaken in vivo. Thus, the aim of this work is to elucidate whether IRE1 RNase activity can also be modeled in cDC1s differentiated in vitro and reveal the functional consequences of such activation in cells stimulated with viral components. Our data show that cultures of optimally differentiated cDC1s recapitulate several features of IRE1 activation noticed in in vivo counterparts and identify the viral analog Poly(I:C) as a potent UPR inducer in the lineage. In vitro differentiated cDC1s display constitutive IRE1 RNase activity and hyperactivate IRE1 RNase upon genetic deletion of XBP1s, which regulates production of the proinflammatory cytokines IL-12p40, TNF-α and IL-6, Ifna and Ifnb upon Poly(I:C) stimulation. Our results show that a strict regulation of the IRE1/XBP1s axis regulates cDC1 activation to viral agonists, expanding the scope of this UPR branch in potential DC-based therapies.

Structural and biochemical characterization of Schlafen11 N-terminal domain.

Schlafen11 (SLFN11) is one of the most studied Schlafen proteins that plays vital roles in cancer therapy and virus-host interactions. Herein, we determined the crystal structure of the Sus scrofa SLFN11 N-terminal domain (NTD) to 2.69 Å resolution. sSLFN11-NTD is a pincer-shaped molecule that shares an overall fold with other SLFN-NTDs but exhibits distinct biochemical characteristics. sSLFN11-NTD is a potent RNase cleaving type I and II tRNAs and rRNAs, and with preference to type II tRNAs. Consistent with the codon usage-based translation suppression activity of SLFN11, sSLFN11-NTD cleaves synonymous serine and leucine tRNAs with different efficiencies in vitro. Mutational analysis revealed key determinates of sSLFN11-NTD nucleolytic activity, including the Connection-loop, active site, and key residues essential for substrate recognition, among which E42 constrains sSLFN11-NTD RNase activity, and all nonconservative mutations of E42 stimulated RNase activities. sSLFN11 inhibited the translation of proteins with a low codon adaptation index in cells, which mainly dependent on the RNase activity of the NTD because E42A enhanced the inhibitory effect, but E209A abolished inhibition. Our findings provide structural characterization of an important SLFN11 protein and expand our understanding of the Schlafen family.

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium.

Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

ISG20 stimulates anti-tumor immunity via a double-stranded RNA-induced interferon response in ovarian cancer.

Augmentation of endogenous double-stranded RNA (dsRNA) has become a promising strategy for activating anti-tumor immunity through induction of type I interferon (IFN) in the treatment of ovarian carcinoma. However, the underlying regulatory mechanisms of dsRNA in ovarian carcinoma remain elusive. From The Cancer Genome Atlas (TCGA), we downloaded RNA expression profiles and clinical data of patients with ovarian carcinoma. Using the consensus clustering method, patients can be classified by their expression level of core interferon-stimulated genes (ISGs): IFN signatures high and IFN signatures low. The IFN signatures high group had a good prognosis. Gene set enrichment analysis (GSEA) showed that differentially expressed genes (DEGs) were primarily associated with anti-foreign immune responses. Based on results from protein-protein interaction (PPI) networks and survival analysis, ISG20 was identified as a key gene involved in host anti-tumor immune response. Further, elevated ISG20 expression in ovarian cancer cells led to increased IFN-β production. The elevated interferon improved the immunogenicity of tumor cells and generated chemokines that attract immune cells to infiltrate the area. Upon overexpression of ISG20, endogenous dsRNA accumulated in the cell and stimulated IFN-β production through the Retinoic acid-inducible gene I (RIG-I)-mediated dsRNA sense pathway. The accumulation of dsRNA was associated with the ribonuclease activity of ISG20. This study suggests that targeting ISG20 is a potential immune therapeutic approach to treat ovarian cancer.

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.

CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.

Domain swapping of the C-terminal helix promotes the dimerization of a novel ribonuclease protein from Mycobacterium tuberculosis.

Polyketide metabolism-associated proteins in Mycobacterium tuberculosis play an essential role in the survival of the bacterium, which makes them potential drug targets for the treatment of tuberculosis (TB). The novel ribonuclease protein Rv1546 is predicted to be a member of the steroidogenic acute regulatory protein-related lipid-transfer (START) domain superfamily, which comprises bacterial polyketide aromatase/cyclases (ARO/CYCs). Here, we determined the crystal structure of Rv1546 in a V-shaped dimer. The Rv1546 monomer consists of four α-helices and seven antiparallel β-strands. Interestingly, in the dimeric state, Rv1546 forms a helix-grip fold, which is present in START domain proteins, via three-dimensional domain swapping. Structural analysis revealed that the conformational change of the C-terminal α-helix of Rv1546 might contribute to the unique dimer structure. Site-directed mutagenesis followed by in vitro ribonuclease activity assays was performed to identify catalytic sites of the protein. This experiment suggested that surface residues R63, K84, K88, and R113 are important in the ribonuclease function of Rv1546. In summary, this study presents the structural and functional characterization of Rv1546 and supplies new perspectives for exploiting Rv1546 as a novel drug target for TB treatment.