Non-coding RNAs (ncRNAs) comprise a set of abundant and functionally diverse RNA molecules. Since the discovery of the first ncRNA in the 1960s, ncRNAs have been shown to be involved in nearly all steps of the central dogma of molecular biology. In recent years, the pace of discovery of novel ncRNAs and their cellular roles has been greatly accelerated by high-throughput sequencing. Advances in sequencing technology, library preparation protocols as well as computational biology helped to greatly expand our knowledge of which ncRNAs exist throughout the kingdoms of life. Moreover, RNA sequencing revealed crucial roles of many ncRNAs in human health and disease. In this review, we discuss the most recent methodological advancements in the rapidly evolving field of high-throughput sequencing and how it has greatly expanded our understanding of ncRNA biology across a large number of different organisms.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Necroptosis facilitates cell death in a controlled manner and is employed by many cell types following injury. It plays a significant role in various liver diseases, albeit the cell-type-specific regulation of necroptosis in the liver and especially hepatocytes, has not yet been conceptualized. We demonstrate that DNA methylation suppresses RIPK3 expression in human hepatocytes and HepG2 cells. In diseases leading to cholestasis, the RIPK3 expression is induced in mice and humans in a cell-type-specific manner. Overexpression of RIPK3 in HepG2 cells leads to RIPK3 activation by phosphorylation and cell death, further modulated by different bile acids. Additionally, bile acids and RIPK3 activation further facilitate JNK phosphorylation, IL-8 expression, and its release. This suggests that hepatocytes suppress RIPK3 expression to protect themselves from necroptosis and cytokine release induced by bile acid and RIPK3. In chronic liver diseases associated with cholestasis, induction of RIPK3 expression may be an early event signaling danger and repair through releasing IL-8.

Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.

Clouds have been regarded as atmospheric oasis for microbes including psychrophilic bacteria (Delort et al., 2017; Péguilhan et al., 2023). However, adaptations of bacteria to the cloud environment and interactions with viruses are not fully understood. In this study, cloudwater was sampled with six compact Caltech active strand cloud water collectors (Demoz et al., 1996) on the Mount Verde, a mountain of 744 m height on the São Vicente island in the tropical Atlantic Ocean (van Pinxteren et al., 2020) and stored frozen. From iron-flocculated and filtered cloudwater samples, DNA was short-read sequenced for metagenomics, and 24 bacteria were additionally isolated from these samples on Luria-Bertani (LB) and Reasoner's 2A (R2A) agar. After purification, the bacterial DNA was subjected to whole-genome sequencing, revealing a diverse array of microbial taxa. The isolate genomes were identified as belonging to Gram-positive species, including Agrococcus sp., Alkalihalobacillus_A gibsonii_A, Arthrobacter sp., Bacillus spizizenii, Cytobacillus oceanisediminis, Curtobacterium spp., Deinococcus sp., Micrococcus luteus, and Rossellomorea spp., as well as Gram-negative species such as Paracoccus marcusii, and Sphingomonas sp. This microbial diversity highlights the presence of spore-forming, halotolerant, and marine-associated bacteria in cloudwater. The genomes had an average GC content of 58.3% (range 41% – 73%) and encoded for cold-shock genes probably supporting survival during sample freezing and in supercooled cloudwater. The presence of 24 prophages and a diverse arsenal of antiviral defense systems, including adaptive CRISPR immunity targeting viral operational taxonomic units (vOTUs), indicates ongoing bacterial-viral interactions in cloudwater. On average, bacterial strains encoded for five defense systems, with restriction-modification systems being the most common. Interestingly, the isolated strain Sphingomonas sp. MPC37 encoded for the highest number of defense systems (12), indicating its potential ecological significance in this unique environment. Metagenomic sequencing identified 458 vOTUs, with major bacterial hosts predicted as Sphingomonas spp. (75 vOTUs), Deinococcus spp. (15), Novosphingobium spp. (14), and Methylobacterium spp. (13). Analysis of air mass trajectories for the cloudwater suggests a marine origin for certain samples, which were associated with the highest counts of both unique and total vOTUs. We also find genetic variability within a population of closely related viruses (microdiversity). Viral variants arise sequentially during different cloud events and are shared among temporally proximate events. Our results reveal clouds as dynamic microbial and viral ecosystems with complex survival strategies and interactions.ReferencesDelort, A. M., Vaïtilingom, M., Joly, M., … & Deguillaume, L. (2017). Clouds: a transient and stressing habitat for microorganisms. Microbial ecology of extreme environments, 215-245. https://doi.org/10.1007/978-3-319-51686-8_10Demoz, B. B., Collett, J. L., & Daube, B. C. (1996). On the Caltech Active Strand Cloudwater Collectors. Atmospheric Research, 41(1), 47-62. https://doi.org/10.1016/0169-8095(95)00044-5Péguilhan, R., Rossi, F., Joly, M., … & Amato, P. (2023). Clouds, oases for airborne microbes – Differential metagenomics/ metatranscriptomics analyses of cloudy and clear atmospheric situations. bioRxiv, 2023.2012.2014.571671. https://doi.org/10.1101/2023.12.14.571671van Pinxteren, M., Fomba, K. W., Triesch, N., . . . & Herrmann, H. (2020). Marine organic matter in the remote environment of the Cape Verde islands – an introduction and overview to the MarParCloud campaign. Atmos. Chem. Phys., 20(11), 6921-6951. https://doi.org/10.5194/acp-20-6921-2020

Background: Cloud water harbors diverse microbial communities despite its extreme oligotrophic conditions. However, the ecological and evolutionary dynamics of viruses in these transient atmospheric habitats remain poorly understood. Clouds have traditionally been regarded primarily as passive carriers of microorganisms rather than as active ecological environments supporting microbial interactions. In this study, cloud water was sampled at Mount Verde, Cape Verde Islands (744 m a.s.l.). We performed metagenomic analyses of iron-flocculated cloud water alongside genome analyses of a bacterial isolate and metagenome-assembled genomes using established bioinformatic approaches. Viral diversity, virus-host interactions, metabolic functions, genetic adaptations, and viral population dynamics across cloud events were investigated. In addition, UV-B resistance experiments were conducted for a novel cloud-water isolate. Results: We isolated 24 cloud water bacteria, including four novel species lineages, and recovered 62 high-quality metagenome-assembled genomes, including 10 novel species lineages. We identified 458 viral operational taxonomic units and 237 virus-host linkages across diverse prokaryotic hosts, revealing active viral predation across diverse bacterial taxa. In addition, CRISPR spacer matches from isolates of novel bacterial lineages such as Deinococcus nubigenus MPC36 were found. Viruses carried genes involved in host adaptation to environmental stressors, including cold-shock response, UV radiation resistance, and osmotic stress. In addition, viral populations exhibited SNP-level microdiversity and shifts in single-nucleotide variant composition across temporally proximate cloud events, indicating rapid population turnover. Experimental characterization of the cloud isolate Curtobacterium nubigenum MPC39 further revealed pronounced resistance to UV-B radiation and the presence of an inducible prophage, Curtobacterium phage vB_CnuS_Cirrus1 assigned to the new viral family Nebulaviridae , which could be validated in transmission electron microscopy. Reconstructed genomes from cloud-associated bacteria encoded carbon monoxide dehydrogenase genes and UV resistance genes, suggesting trace gas metabolism and enhanced UV protection as survival strategies in oligotrophic cloud droplets. In silico replication rates estimated using iRep were consistent with active bacterial replication at the time of sampling. Conclusions: Together, these findings demonstrate that clouds are not merely passive carriers of microorganisms, but dynamic atmospheric ecosystems in which virus-host interactions shape microbial diversity and contribute to microbial turnover, atmospheric dispersal, and cloud-water biogeochemistry.