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RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell–cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse β-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host–parasite or symbiotic interactions.

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter ).

COVID-19 is a disease caused by a virus named SARS-CoV-2. SARS-CoV-2 is a single-stranded positive-sense RNA virus. Reverse transcription quantitative PCR (RT–qPCR) assays are the gold standard molecular test for detection of RNA viruses. The aim of this study was to construct an RNA-positive control based on MS2 phage-like particles (MS2 VLPs) to detect SARS-CoV-2 RNA. pCDFDuet-1 was used as a one-plasmid double-expression system to construct MS2 VLPs containing ssRNA of SARS-CoV-2. The sequence encoding one copy of maturase, His-tag and coat protein dimer was cloned and inserted into MCS1 of the plasmid; the fragment encoding protein N and ORF1ab from SARS-CoV-2 was cloned and inserted into MCS2. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and expression of the construct was induced by 1 mM isopropyl-L-thio-D-galactopyranoside (IPTG) at 30°C for 12 hours. MS2 VLPs were purified and collected with Ni-NTA affinity chromatography columns. The size and shape of the MS2 VLPs were verified by transmission electron microscopy, and the stability of MS2 VLP packaged RNA was evaluated by treatment with RNase A. Effects of storage temperature and buffer on MS2 VLP stability were also investigated. The results showed that SARS-CoV-2 MS2 VLPs could be successfully produced by this one-plasmid double-expression system. MS2 VLPs showed high stability and may be used as a positive control in molecular diagnosis of COVID-19.

In this work, the issue of hospital and urban wastewater treatment is studied in two different contexts, in Switzerland and in developing countries (Ivory Coast and Colombia). For this purpose, the treatment of municipal wastewater effluents is studied, simulating the developed countries’ context, while cheap and sustainable solutions are proposed for the developing countries, to form a barrier between effluents and receiving water bodies. In order to propose proper methods for each case, the characteristics of the matrices and the targets are described here in detail. In both contexts, the use of Advanced Oxidation Processes (AOPs) is implemented, focusing on UV-based and solar-supported ones, in the respective target areas. A list of emerging contaminants and bacteria are firstly studied to provide operational and engineering details on their removal by AOPs. Fundamental mechanistic insights are also provided on the degradation of the effluent wastewater organic matter. The use of viruses and yeasts as potential model pathogens is also accounted for, treated by the photo-Fenton process. In addition, two pharmaceutically active compound (PhAC) models of hospital and/or industrial origin are studied in wastewater and urine, treated by all accounted AOPs, as a proposed method to effectively control concentrated point-source pollution from hospital wastewaters. Their elimination was modeled and the degradation pathway was elucidated by the use of state-of-the-art analytical techniques. In conclusion, the use of light-supported AOPs was proven to be effective in degrading the respective target and further insights were provided by each application, which could facilitate their divulgation and potential application in the field.

Transcription, RNA processing, and chromatin‐related factors all interact with RNA polymerase II (Pol II) to ensure proper timing and coordination of transcription and co‐transcriptional processes. Many transcription elongation regulators must function simultaneously to coordinate these processes, yet few strategies exist to explore the complement of factors regulating specific stages of transcription. To this end, we developed a strategy to purify Pol II elongation complexes from subgenic regions of a single gene, namely the 5′ and 3′ regions, using sequences in the nascent RNA. Applying this strategy to Saccharomyces cerevisiae, we determined the specific set of factors that interact with Pol II at precise stages during transcription. We identify many known region‐specific factors as well as determine unappreciated associations of regulatory factors during early and late stages of transcription. These data reveal a role for the transcription termination factor, Rai1, in regulating the early stages of transcription genome‐wide and support the role of Bye1 as a negative regulator of early elongation. We also demonstrate a role for the ubiquitin ligase, Bre1, in regulating Pol II dynamics during the latter stages of transcription. These data and our approach to analyze subgenic transcription elongation complexes will shed new light on the myriad factors that regulate the different stages of transcription and coordinate co‐transcriptional processes. Synopsis Subgenic isolation of RNA polymerase II transcription elongation complexes reveals unique 5′ and 3′ elongation complexes and uncovers roles for Rai1 and Bye1 in regulating early elongation and Bre1 in regulating late‐stage elongation. A strategy is presented for subgenic isolation of transcription elongation complexes. Unique transcription elongation complexes are identified at 5′ and 3′ regions of a single gene. Bye1 and Rai1 regulate early transcription elongation. Bre1 regulates transcriptional pausing at the 3′ end of genes. Graphical Abstract Subgenic isolation of RNA polymerase II transcription elongation complexes reveals unique 5′ and 3′ elongation complexes and uncovers roles for Rai1 and Bye1 in regulating early elongation and Bre1 in regulating late‐stage elongation.

Bacteriophage-based applications have a renaissance today, increasingly marking their use in industry, medicine, food processing, biotechnology, and more. However, phages are considered resistant to various harsh environmental conditions; besides, they are characterized by high intra-group variability. Phage-related contaminations may therefore pose new challenges in the future due to the wider use of phages in industry and health care. Therefore, in this review, we summarize the current knowledge of bacteriophage disinfection methods, as well as highlight new technologies and approaches. We discuss the need for systematic solutions to improve bacteriophage control, taking into account their structural and environmental diversity.

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then theMat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

Sanguisorba tannins are the major active ingredients in Sanguisorba ofJicinalis L. (Rosaceae), one of the most popular herbal medicines in China, is widely prescribed for hemostasis. In this study, three kinds of tannins extract from Sanguisorba officinalis L. (Rosaceae), and the metabolites in vivo and in vitro were detected and identified by high-pressure liquid chromatography, coupled with linear ion trap orbitrap tandem mass spectrometry (HPLC–LTQ–Orbitrap). For in vivo assessment, the rats were administered at a single dose of 150 mg/kg, after which 12 metabolites were found in urine, 6 metabolites were found in feces, and 8 metabolites were found in bile, while metabolites were barely found in plasma and tissues. For in vitro assessment, 100 μM Sanguisorba tannins were incubated with rat liver microsomes, liver cytosol, and feces, after which nine metabolites were found in intestinal microbiota and five metabolites were found in liver microsomes and liver cytosol. Moreover, the metabolic pathways of Sanguisorba tannins were proposed, which shed light on their mechanism.

The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs in planta. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach’s potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.Key messageWe have established a fast and straightforward editing-based assay to analyse RBP/RNA interactions, demonstrating PTB binding to an intronic sequence adjacent to a cassette exon as prerequisite for its inclusion.

Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general biophysical properties impact loading and delivery of RNA by EVs, we developed a platform for actively loading engineered cargo RNAs into EVs. In our system, the MS2 bacteriophage coat protein was fused to EV-associated proteins, and the cognate MS2 stem loop was engineered into cargo RNAs. Using this Targeted and Modular EV Loading (TAMEL) approach, we identified a configuration that substantially enhanced cargo RNA loading (up to 6-fold) into EVs. When applied to vesicles expressing the vesicular stomatitis virus glycoprotein (VSVG) - gesicles - we observed a 40-fold enrichment in cargo RNA loading. While active loading of mRNA-length (>1.5 kb) cargo molecules was possible, active loading was much more efficient for smaller (~0.5 kb) RNA molecules. We next leveraged the TAMEL platform to elucidate the limiting steps in EV-mediated delivery of mRNA and protein to prostate cancer cells, as a model system. Overall, most cargo was rapidly degraded in recipient cells, despite high EV-loading efficiencies and substantial EV uptake by recipient cells. While gesicles were efficiently internalized via a VSVG-mediated mechanism, most cargo molecules were rapidly degraded. Thus, in this model system, inefficient endosomal fusion or escape likely represents a limiting barrier to EV-mediated transfer. Altogether, the TAMEL platform enabled a comparative analysis elucidating a key opportunity for enhancing EV-mediated delivery to prostate cancer cells, and this technology should be of general utility for investigations and applications of EV-mediated transfer in other systems.