DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing.

By temporarily distorting the DNA double helix, the moving RNA polymerases can lead to the formation of non-B DNA structures. One of the most abundant and largest non-B DNA structures in the genome is the R-loop, a three-stranded structure forming when the nascent RNA hybridizes with its DNA template, thereby extruding the non-template DNA strand. Growing evidence suggests that at least a subset of R-loops could induce transcription stress and genome instability, although the direct, primary consequences of R-loop formation on the surrounding chromatin are still unclear.To understand the direct impact of R-loops on transcription and genome stability, accurate and quantitative mapping of R-loops is essential. R-loop mapping is commonly achieved using the antibody-based DNA:RNA Immunoprecipitation (DRIP) strategy. While it is reasonably straightforward to obtain robust DRIP enrichments from human cells, this has proved harder in yeast, where DRIP signals are often relatively weak, with a poor signal-to-noise ratio. Although it is unclear whether such weak signals stem from a technical or a biological reality, they make the accurate quantification of DRIP signals all the more important, especially when deep sequencing is used to monitor and quantify the distribution of R-loops genome-wide. Here we propose a DRIP protocol that has been optimized for the mapping and the quantification of R-loops in Schizosaccharomyces pombe but that can also be used in Saccharomyces cerevisiae. As a result, this protocol can be used to generate calibrated DRIP-seq data, where genomic DNA extracted from S. cerevisiae serves as spike-in reference.

Aqueous suspensions of Fuller's earth potentiate the adsorption capacities of paraoxon and improve skin decontamination properties.

Fuller's earth (FE) is a phyllosilicate used as a powder for household or skin decontamination due to its adsorbent properties. Recent studies have shown that water suspensions exhibit similar adsorbent capacities. FE is heterogeneous due to its composition of elementary clay aggregates and heavy metal particles. Here, FE toxicity was assessed in vitro on skin cells and in vivo on Danio rerio embryos. Among the suspensions tested (5%, 9.1% and 15% w/w), only the highest one shows weak toxicity. Suspensions were tested for ex vivo dermal decontamination into pig ear skin and human abdominal skin using diffusion cells and paraoxon as organophosphorus contaminant. After 24 h of diffusion, no difference was observed in the paraoxon concentration in the receptor compartment whether the decontamination was carried out with FE in powder or in suspension form. In presence of FE suspensions, we observed the disappearance of paraoxon from the stratum corneum, the reservoir compartment, independently of the suspensions' concentration. We suggest that water potentiates the absorbing capacities of FE powder by intercalating between clay lamellas leading to the appearance of new adsorption zones and swelling. These data support the use of FE aqueous suspensions as a safe tool for organophosphorus skin decontamination.

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA.

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5' untranslated region (UTR) and two 3' UTRs of the human ß-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5' UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5'PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5' UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes.

The mechanisms leading to the accumulation of the SMC complexes condensins around specific transcription units remain unclear. Observations made in bacteria suggested that RNA polymerases (RNAPs) constitute an obstacle to SMC translocation, particularly when RNAP and SMC travel in opposite directions. Here we show in fission yeast that gene termini harbour intrinsic condensin-accumulating features whatever the orientation of transcription, which we attribute to the frequent backtracking of RNAP at gene ends. Consistent with this, to relocate backtracked RNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation of condensin at gene ends and to redistribute it evenly within transcription units, indicating that RNAP backtracking may play a key role in positioning condensin. Formalization of this hypothesis in a mathematical model suggests that the inclusion of a sub-population of RNAP with longer dwell-times is essential to fully recapitulate the distribution profiles of condensin around active genes. Taken together, our data strengthen the idea that dense arrays of proteins tightly bound to DNA alter the distribution of condensin on chromosomes.

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.

Transcriptional induction of cell wall remodelling genes is coupled to microtubule-driven growth isotropy at the shoot apex in Arabidopsis.

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.