The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

Investigation of RNA metabolism through large-scale genetic interaction profiling in yeast.

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.

SHAMAN: a user-friendly website for metataxonomic analysis from raw reads to statistical analysis.

BACKGROUND: Comparing the composition of microbial communities among groups of interest (e.g., patients vs healthy individuals) is a central aspect in microbiome research. It typically involves sequencing, data processing, statistical analysis and graphical display. Such an analysis is normally obtained by using a set of different applications that require specific expertise for installation, data processing and in some cases, programming skills. RESULTS: Here, we present SHAMAN, an interactive web application we developed in order to facilitate the use of (i) a bioinformatic workflow for metataxonomic analysis, (ii) a reliable statistical modelling and (iii) to provide the largest panel of interactive visualizations among the applications that are currently available. SHAMAN is specifically designed for non-expert users. A strong benefit is to use an integrated version of the different analytic steps underlying a proper metagenomic analysis. The application is freely accessible at http://shaman.pasteur.fr/ , and may also work as a standalone application with a Docker container (aghozlane/shaman), conda and R. The source code is written in R and is available at https://github.com/aghozlane/shaman . Using two different datasets (a mock community sequencing and a published 16S rRNA metagenomic data), we illustrate the strengths of SHAMAN in quickly performing a complete metataxonomic analysis. CONCLUSIONS: With SHAMAN, we aim at providing the scientific community with a platform that simplifies reproducible quantitative analysis of metagenomic data.

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans.

Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5' ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans. These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

Identification of Links Between Cellular Pathways by Genetic Interaction Mapping (GIM).

The yeast systematic deletion collection offered the basis for a number of different strategies that establish functional links between genes by analyzing the phenotype of cells that combine two different deletions or mutations. A distinguishing feature of the collection is the presence of molecular barcodes at each deleted locus, which can be used to quantify the presence and abundance of cells bearing a given allele in a complex mix. As a result, a large number of mutants can be tested in batch cultures, replacing tedious manipulation of thousands of individual strains with a barcode microarray readout. Barcode-based genetic screens like Genetic Interaction Mapping (GIM) thus require little investment in terms of specific equipment, are fast to perform, and allow precise measurements of double mutant growth rates for both aggravating (synthetic sick) and alleviating (epistatic) effects. We describe here protocols for preparing the pools of haploid double mutant S. cerevisiae cells, testing their composition with barcode microarrays, and analyzing the results to extract useful functional information.

Quality control of transcription start site selection by nonsense-mediated-mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA quality-control pathway targeting transcripts such as messenger RNAs harboring premature stop-codons or short upstream open reading frame (uORFs). Our transcription start sites (TSSs) analysis of Saccharomyces cerevisiae cells deficient for RNA degradation pathways revealed that about half of the pervasive transcripts are degraded by NMD, which provides a fail-safe mechanism to remove spurious transcripts that escaped degradation in the nucleus. Moreover, we found that the low specificity of RNA polymerase II TSSs selection generates, for 47% of the expressed genes, NMD-sensitive transcript isoforms carrying uORFs or starting downstream of the ATG START codon. Despite the low abundance of this last category of isoforms, their presence seems to constrain genomic sequences, as suggested by the significant bias against in-frame ATGs specifically found at the beginning of the corresponding genes and reflected by a depletion of methionines in the N-terminus of the encoded proteins.

Long open reading frame transcripts escape nonsense-mediated mRNA decay in yeast.

Nonsense-mediated mRNA decay (NMD) destabilizes eukaryotic transcripts with long 3' UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF) regions and with the same long, destabilizing 3' UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3' UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3' UTR, short translation length is an important feature of NMD substrates in yeast.

Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products.

Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.

Sodium selenide toxicity is mediated by O2-dependent DNA breaks.

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (•)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.

The yeast RPL9B gene is regulated by modulation between two modes of transcription termination.

RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.

Genoscape: a Cytoscape plug-in to automate the retrieval and integration of gene expression data and molecular networks.

UNLABELLED: Genoscape is an open-source Cytoscape plug-in that visually integrates gene expression data sets from GenoScript, a transcriptomic database, and KEGG pathways into Cytoscape networks. The generated visualisation highlights gene expression changes and their statistical significance. The plug-in also allows one to browse GenoScript or import transcriptomic data from other sources through tab-separated text files. Genoscape has been successfully used by researchers to investigate the results of gene expression profiling experiments. AVAILABILITY: Genoscape is an open-source software freely available from the Genoscape webpage (http://www.pasteur.fr/recherche/unites/Gim/genoscape/). Installation instructions and tutorial can also be found at this URL.

Widespread bidirectional promoters are the major source of cryptic transcripts in yeast.

Pervasive and hidden transcription is widespread in eukaryotes, but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae. These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 3' Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis genes, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

Application of support vector machines to 1H NMR data of fish oils: methodology for the confirmation of wild and farmed salmon and their origins.

Support vector machines (SVMs) were used as a novel learning machine in the authentication of the origin of salmon. SVMs have the advantage of relying on a well-developed theory and have already proved to be successful in a number of practical applications. This paper provides a new and effective method for the discrimination between wild and farm salmon and eliminates the possibility of fraud through misrepresentation of the country of origin of salmon. The method requires a very simple sample preparation of the fish oils extracted from the white muscle of salmon samples. (1)H NMR spectroscopic analysis provides data that is very informative for analysing the fatty acid constituents of the fish oils. The SVM has been able to distinguish correctly between the wild and farmed salmon; however ca. 5% of the country of origins were misclassified.