Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5′-half molecules of tRNA ^HisGUG (the 5′-tRNA ^HisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5′-tRNA half molecules and identified the 5′-tRNA ^ValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5′-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5′-tRNA ^ValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis . The 5′-tRNA ^ValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5′-tRNA ^ValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α′ ₂ , α ₄ and α ₂ β ₂ ) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum , referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε ₂ homodimer and has broad substrate specificity like the α ₂ β ₂ type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε ₂ homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (α ^N , α and β ^C ) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε ₂ and α ₂ β ₂ EndAs were separately acquired through different evolutionary processes.

Inflammation and neonatal hypoxia-ischemia (HI) are important etiological factors of perinatal brain injury. However, underlying mechanisms remain unclear. Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases. Sirtuin-6 is thought to regulate inflammatory and oxidative pathways, such as the extracellular release of the alarmin high mobility group box-1 (HMGB1). The expression and role of sirtuin-6 in neonatal brain injury are unknown. In a well-established model of neonatal brain injury, which encompasses inflammation (lipopolysaccharide, LPS) and hypoxia-ischemia (LPS+HI), we investigated the protein expression of sirtuin-6 and HMGB1, as well as thiol oxidation. Furthermore, we assessed the effect of the antioxidant N-acetyl cysteine (NAC) on sirtuin-6 expression, nuclear to cytoplasmic translocation, and release of HMGB1 in the brain and blood thiol oxidation after LPS+HI. We demonstrate reduced expression of sirtuin-6 and increased release of HMGB1 in injured hippocampus after LPS+HI. NAC treatment restored sirtuin-6 protein levels, which was associated with reduced extracellular HMGB1 release and reduced thiol oxidation in the blood. The study suggests that early reduction in sirtuin-6 is associated with HMGB1 release, which may contribute to neonatal brain injury, and that antioxidant treatment is beneficial for the alleviation of these injurious mechanisms.

The purpose of this paper is to investigate the effects of the touching manners and the motion directions of human finger in recognizing fine surface texture. The authors developed a measurement system to present a step-height of 10 to 1000 mum to the finger of the human subject to measure the human tactile sensation capability. The presentation device can control four parameters of the presentation, which are the step-height, the presentation velocity, the presentation angle, and the presentation temperature. Human subjects actively and passively touched and distinguished the step-heights to determine the different thresholds for step-heights. Also they passively touched and distinguished the step-heights with different motion directions of their fingers to determine the difference thresholds. As the results of the psychophysical experiments, it was found that the distinctive sensitivities of human tactile sensation in active-touch and passive-touch manners are different in discriminating between fine step-heights and that the directions of finger motion have little effect on the human tactile recognition of fine step-heights.

The RNase A superfamily, one of the most extensively studied enzyme families, remains incompletely defined with respect to its endogenous RNA substrates, recognition mechanisms, and physiological functions. Here, we identify eosinophil-derived neurotoxin (EDN/RNase 2) as a principal endoribonuclease mediating tRNA cleavage in the asthmatic lung. Inhalation of house dust mite (HDM) in mice induces robust accumulation of tRNA halves in the lung, coinciding with exclusive upregulation of EDN, but not other ribonucleases. In human lung epithelial cells, internalized EDN cleaves specific tRNAs and generates multiple species of tRNA halves, including immunostimulatory species that activate Toll-like receptor 7 (TLR7). EDN also promotes the release of extracellular vesicles enriched in these RNAs, implicating them in cytokine production during asthma pathogenesis. Biochemical analyses and structural simulations reveal that EDN recognizes the anticodon loop of tRNA by anchoring the phosphate backbones flanking the cleavage site, thereby precisely positioning the C34–A35 phosphodiester bond for catalysis. Furthermore, the highly efficient cleavage by EDN is mediated by conserved elements, including Arg36 in EDN and U33 and C38 in the target tRNA. Together, these findings establish the endogenous RNA substrate of EDN and uncover a molecular mechanism that links RNase A superfamily activity to immune response.