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microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Small RNAs and their associated RNA interference (RNAi) pathways underpin diverse mechanisms of gene regulation and genome defense across all three kingdoms of life and are integral to virus-host interactions. In plants, fungi and many animals, an ancestral RNAi pathway exists as a host defense mechanism whereby viral double-stranded RNA is processed to small RNAs that enable recognition and degradation of the virus. While this antiviral RNAi pathway is not generally thought to be present in mammals, other RNAi mechanisms can influence infection through both viral- and host-derived small RNAs. Furthermore, a burgeoning body of data suggests that small RNAs in mammals can function in a non-cell autonomous manner to play various roles in cell-to-cell communication and disease through their transport in extracellular vesicles. While vesicular small RNAs have not been proposed as an antiviral defense pathway per se, there is increasing evidence that the export of host- or viral-derived RNAs from infected cells can influence various aspects of the infection process. This review discusses the current knowledge of extracellular RNA functions in viral infection and the technical challenges surrounding this field of research. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action., (© 2019 The Authors. WIREs RNA published by Wiley Periodicals, Inc.)

In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The majority of antiviral therapeutics target conserved viral proteins, however, this approach confers selective pressure on the virus and increases the probability of antiviral drug resistance. An alternative therapeutic strategy is to target the host-encoded factors that are required for virus infection, thus minimizing the opportunity for viral mutations that escape drug activity. MicroRNAs (miRNAs) are small noncoding RNAs that play diverse roles in normal and disease biology, and they generally operate through the post-transcriptional regulation of mRNA targets. We have previously identified cellular miRNAs that have antiviral activity against a broad range of herpesvirus infections, and here we extend the antiviral profile of a number of these miRNAs against influenza and respiratory syncytial virus. From these screening experiments, we identified broad-spectrum antiviral miRNAs that caused >75% viral suppression in all strains tested, and we examined their mechanism of action using reverse-phase protein array analysis. Targets of lead candidates, miR-124, miR-24, and miR-744, were identified within the p38 mitogen-activated protein kinase (MAPK) signaling pathway, and this work identified MAPK-activated protein kinase 2 as a broad-spectrum antiviral target required for both influenza and respiratory syncytial virus (RSV) infection., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Optical metrology systems crucially rely on the dimensional stability of the optical path between their individual optical components. We present in this paper a novel adhesive bonding technology for setup of quasi-monolithic systems and compare selected characteristics to the well-established state-of-the-art technique of hydroxide-catalysis bonding. It is demonstrated that within the measurement resolution of our ultraprecise custom heterodyne interferometer, both techniques achieve an equivalent passive path length and tilt stability for time scales between 0.1 mHz and 1 Hz. Furthermore, the robustness of the adhesive bonds against mechanical and thermal inputs has been tested, making this new bonding technique in particular a potential option for interferometric applications in future space missions. The integration process itself is eased by long time scales for alignment, as well as short curing times.

I examined the aerobic trunk muscles, which are used for call production, of male frogs from species that breed in different thermal environments to test the hypothesis that cold-adapted frogs should have fewer capillaries per unit mitochondrial volume in oxidative muscles than warm-adapted frogs because of reduced mitochondrial function at low temperatures. The species of interest were the cold-temperate Pseudacris crucifer and the warm-tropical Hyla microcephala in the family Hylidae, and the cold-temperate Rana sylvatica and the warm-temperate Rana clamitans in the family Ranidae. Trunk-muscle mitochondrial volume, V(V)(mt,f), was proportionally higher in species with higher mean calling rates (number of notes per hour), irrespective of the familial affinity of a species and the thermal environment in which it vocalized. Trunk-muscle capillary length density, J(V)(c,f), was significantly lower in P. crucifer than in H. microcephala because of significantly higher mean fiber area, a macro (f). Conversely, trunk-muscle J(V)(c,f) was similar in the two ranid species. Using total capillary length, J(c), and total mitochondrial volume, V(mt,m), as a measure of maximal oxygen supply and demand, respectively, in trunk muscles, J(c)-to-V(mt,m) ratios were significantly lower in cold-adapted P. crucifer (4.3 km cm(-3)) and R. sylvatica (4.8 km cm(-3)) than in warm-adapted H. microcephala (7.1 km cm(-3)) and R. clamitans (6.4 km cm(-3)). In contrast, J(c)-to-V(mt,m) ratios in the more anaerobic gastrocnemius muscle of these species was not related to the thermal environment of a species, which may reflect capillaries conforming to microcirculatory functions, e.g. lactate removal, that take precedence over oxygen delivery. Mitochondrial cristae surface area, S(V)(im,mt), in P. crucifer trunk and gastrocnemius muscles (37.7+/-1.6 and 35.9+/-1.5 m(2 )cm(-3) respectively) was, on average, similar to mammalian values, suggesting equivalent structural capacities of muscle mitochondria in these two taxa. Taken together, the present data suggest that trunk-muscle respiratory design may reflect a capillary supply commensurate with maximal levels of oxygen delivery set by mitochondria operating at different environmental temperatures. P. crucifer and H. microcephala trunk muscles were also characterized by a high lipid content, which contrasted with a near absence of trunk-muscle lipids in R. sylvatica and R. clamitans. The extraordinarily high lipid content of P. crucifer trunk muscles (26 % of muscle volume) may serve as an auxiliary oxygen pathway to mitochondria and thus compensate in part for this tissue's reduced capillary/fiber interface. The effect of potentially high depletion rates of trunk-muscle lipid stores on metabolic rates of male frogs while calling is discussed.

Latent infection of the trigeminal ganglion with herpes simplex virus type 2 (HSV-2) was studied in guinea pigs by in situ DNA hybridization. Frozen ganglion sections from animals killed during the period of latent virus infection were studied under nondenaturing conditions. Some sections were treated with deoxyribonuclease (DNase) or ribonuclease (RNase) before incubation with HSV DNA probes. HSV probes consisted of viral DNA nick translated and labeled in vitro with tritiated nucleotides. Bacteriophage lambda DNA, similarly prepared, was used as a control probe. The lambda probe was negative in all situations, including HSV-2-infected monolayer cells in cell culture. HSV-2 probes produced heavy label and, therefore, evidence of hybridization with HSV-2-infected monolayer cells. When HSV-2 probes were incubated with latently infected ganglion sections, hybridization was detected in 71% of guinea pigs and 46% of ganglia. Label was seen only in neurons, and in positive ganglia 0.3 to 5% of neurons were labeled. The amount of label was markedly decreased by pretreatment of ganglion sections with RNase but not DNase, indicating that the DNA probes hybridized to HSV messenger RNA in the latently infected ganglia.