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The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by Arabidopsis Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non-cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

The antimicrobial defense of the skin is partially mediated by RNase 7, an abundant ribonuclease of the stratum corneum (SC). Here, we investigated the expression and regulation of members of the RNase A family and of the endogenous RNase inhibitor (RI) protein in epidermal keratinocytes (KCs). Reverse transcription-PCR screening revealed that KCs expressed not only RNase 7 but also RNase 5, which was shown earlier to kill the yeast Candida albicans, as well as RNase 1, RNase 4, and RI. The mRNA and protein levels of RNase 5, RNase 7, and RI increased during KC differentiation. When RNase 5 and RNase 7 were incubated with RI in vitro, not only their ribonucleolytic activities but also their antimicrobial activities were strongly suppressed. Immunochemical analyses revealed that SC contains RNase 5, whereas RI was not detectable. Unlike recombinant RNase 5, recombinant RI was degraded when exposed to SC extract. The addition of aprotinin prevented the degradation of RI, indicating that serine proteases of the SC cleave RI. Taken together, this study adds RNase 5 to the list of antimicrobial factors present in the SC and suggests that proteases contribute indirectly to the defense function of the SC by releasing the RI-mediated inhibition of RNase 5 and RNase 7.

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins. Responsive hydrogels are of interest for diagnostic and controlled drug release applications. Here, the authors report on the design of a hydrogel with protein triggered release of an embedded protein through recovery of enzyme activity and subsequent degradation of supramolecular fibers.

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.

Included in the armamentarium of antimicrobial defenses of human skin are the RNases. In this issue, Abtin et al. report new details about how this antimicrobial system is deployed. Although present throughout the epidermis, RNases appear to be free to act only within the stratum corneum. Elsewhere in the epidermis RNases are complexed with an inhibitory protein. In the stratum corneum proteases degrade the inhibitor, freeing the RNase and liberating it to function in antimicrobial defense.

The Ribonuclease A Superfamily is composed of a group of structurally similar peptides that are secreted by immune cells and epithelial tissues. Several members of the Ribonuclease A Superfamily demonstrate antimicrobial activity, and it has been suggested that some of these ribonucleases play an essential role in host defense. Ribonuclease 7 (RNase 7) is an epithelial-derived secreted peptide with potent broad-spectrum antimicrobial activity. This review summarizes the published literature on RNase 7’s antimicrobial properties, structure, regulation, and contributions to host defense. In doing so, we conclude by highlighting key knowledge gaps that must be investigated to completely understand the potential of developing RNase 7 as a novel therapeutic for human infectious diseases.

Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-α mRNA through binding to the 3′ untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1β, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of T H17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.

The crowded intracellular environment influences RNA structure and interactions. While in-cell nuclear magnetic resonance (NMR) spectroscopy allows atomic-resolution studies of RNA in living human cells, its application is limited due to rapid RNA degradation. In this study, we introduced an RNase inhibitor cocktail into living human cells and acquired in-cell NMR spectra of an RNA aptamer that strongly binds to HIV-1 Tat, both with and without a peptide derived from Tat. Introduction of the RNase inhibitor cocktail effectively suppressed RNA degradation. The significantly extended lifetime enabled the observation of intact in-cell NMR spectra for both the free aptamer and its complex with the peptide in living human cells at physiological temperatures. The in-cell NMR spectra of the free and complexed forms provided structural insights into RNA in living cells for understanding the mechanism of tight binding, including the formation of U-A-U base triples only in the complex. This was achieved without chemical modifications to the aptamer. The easily applicable and cost-effective nature of the RNase inhibitor cocktail makes this technique suitable for a wide range of in-cell NMR analyses for RNA. This innovation offers a pathway to unravel cellular processes and design novel RNA-focused medications.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.