Alternative splicing induced by bacterial pore-forming toxins sharpens CIRBP-mediated cell response to Listeria infection.

Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based types of regulation, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of 'poison exons'. We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin or aerolysin) led to the dephosphorylation of SRSFs via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.

Coordination of transcriptional and translational regulations in human epithelial cells infected by Listeria monocytogenes.

The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a time-course of infection of human intestinal epithelial cells by an epidemic strain of Listeria monocytogenes, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5'-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (PABPC1) slowed down the intracellular multiplication of Listeria monocytogenes, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.

Extensive degradation of RNA precursors by the exosome in wild-type cells.

The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.

Mammalian microRNAs and long noncoding RNAs in the host-bacterial pathogen crosstalk.

Gene expression regulation is a critical question in host-pathogen interactions, and RNAs act as key players in this process. In this review, we focus on the mammalian RNA response to bacterial infection, with a special interest on microRNAs and long non-coding RNAs. We discuss the role of cellular miRNAs in immunity, the implication of circulating miRNAs as well as the influence of the microbiome on the miRNA response. We also review how pathogens counteract the host miRNA expression. Interestingly, bacterial non-coding RNAs regulate host gene expression and conversely eukaryotic miRNAs may regulate bacterial gene expression. Overall, the characterization of RNA regulatory networks represents an emerging theme in the field of host pathogen interactions.

ALFA: annotation landscape for aligned reads.

BACKGROUND: The last 10 years have seen the rise of countless functional genomics studies based on Next-Generation Sequencing (NGS). In the vast majority of cases, whatever the species, whatever the experiment, the two first steps of data analysis consist of a quality control of the raw reads followed by a mapping of those reads to a reference genome/transcriptome. Subsequent steps then depend on the type of study that is being made. While some tools have been proposed for investigating data quality after the mapping step, there is no commonly adopted framework that would be easy to use and broadly applicable to any NGS data type. RESULTS: We present ALFA, a simple but universal tool that can be used after the mapping step on any kind of NGS experiment data for any organism with available genomic annotations. In a single command line, ALFA can compute and display distribution of reads by categories (exon, intron, UTR, etc.) and biotypes (protein coding, miRNA, etc.) for a given aligned dataset with nucleotide precision. We present applications of ALFA to Ribo-Seq and RNA-Seq on Homo sapiens, CLIP-Seq on Mus musculus, RNA-Seq on Saccharomyces cerevisiae, Bisulfite sequencing on Arabidopsis thaliana and ChIP-Seq on Caenorhabditis elegans. CONCLUSIONS: We show that ALFA provides a powerful and broadly applicable approach for post mapping quality control and to produce a global overview using common or dedicated annotations. It is made available to the community as an easy to install command line tool and from the Galaxy Tool Shed.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression.

The model opportunistic pathogen Listeria monocytogenes has been the object of extensive research, aiming at understanding its ability to colonize diverse environmental niches and animal hosts. Bacterial transcriptomes in various conditions reflect this efficient adaptability. We review here our current knowledge of the mechanisms allowing L. monocytogenes to respond to environmental changes and trigger pathogenicity, with a special focus on RNA-mediated control of gene expression. We highlight how these studies have brought novel concepts in prokaryotic gene regulation, such as the 'excludon' where the 5'-UTR of a messenger also acts as an antisense regulator of an operon transcribed in opposite orientation, or the notion that riboswitches can regulate non-coding RNAs to integrate complex metabolic stimuli into regulatory networks. Overall, the Listeria model exemplifies that fine RNA tuners act together with master regulatory proteins to orchestrate appropriate transcriptional programmes.

Endonucleolytic RNA cleavage by a eukaryotic exosome.

The exosome is a major eukaryotic nuclease located in both the nucleus and the cytoplasm that contributes to the processing, quality control and/or turnover of a large number of cellular RNAs. This large macromolecular assembly has been described as a 3'-->5' exonuclease and shown to contain a nine-subunit ring structure evolutionarily related to archaeal exosome-like complexes and bacterial polynucleotide phosphorylases. Recent results have shown that, unlike its prokaryotic counterparts, the yeast and human ring structures are catalytically inactive. In contrast, the exonucleolytic activity of the yeast exosome core was shown to be mediated by the RNB domain of the eukaryote-specific Dis3 subunit. Here we show, using in vitro assays, that yeast Dis3 has an additional endoribonuclease activity mediated by the PIN domain located at the amino terminus of this multidomain protein. Simultaneous inactivation of the endonucleolytic and exonucleolytic activities of the exosome core generates a synthetic growth phenotype in vivo, supporting a physiological function for the PIN domain. This activity is responsible for the cleavage of some natural exosome substrates, independently of exonucleolytic degradation. In contrast with current models, our results show that eukaryotic exosome cores have both endonucleolytic and exonucleolytic activities, mediated by two distinct domains of the Dis3 subunit. The mode of action of eukaryotic exosome cores in RNA processing and degradation should be reconsidered, taking into account the cooperation between its multiple ribonucleolytic activities.

Regulation of cold-inducible RNA-binding protein (CIRBP) in response to cellular stresses.

Cold-inducible RNA-Binding Protein (CIRBP) is a general stress-response factor in vertebrates harboring two domains: an RNA-recognition motif and a regulatory domain rich in RG/RGG motifs. CIRBP has been described to bind mRNAs upon various stress conditions (cold, infections, UV, hypoxia …) and regulate their stability and translation. The proteins encoded by its targets are involved in key stress-responsive cellular pathways including apoptosis, inflammation, cell proliferation or translation, thus allowing their coordination. Due to its role in regulating central cellular functions, the expression of CIRBP is tightly controlled. We review here current understanding of the multiple mechanistic layers affecting CIRBP expression and function. Beyond transcriptional regulation by cold-responsive elements and the use of alternative promoters and transcription start sites, CIRBP undergoes various alternative splicing (AS) events which, depending on conditions, modulate the stability of CIRBP transcripts and/or impact the sequence of the encoded polypeptide. Typically, whilst CIRBP expression is induced in the context of hypothermia or viral infection, AS events preferentially address alternative isoforms towards mRNA degradation pathways in response to heat stress or to bacterial-secreted pore forming toxins. Post-translational modifications of CIRBP, mostly in its RGG domain, also condition CIRBP subcellular localization and access to its targets, thereby promoting or inhibiting their expression. For instance, phosphorylation and methylation events gate CIRBP nuclear to cytoplasmic translocation and control its recruitment to stress granules. Considering the therapeutic potential of modulating the expression and function of this central player in stress responses, a fine understanding of CIRBP regulation mechanisms deserves further attention.

At the crossroads of epidemiology and biology: Bridging the gap between SARS-CoV-2 viral strain properties and epidemic wave characteristics.

The COVID-19 pandemic has given rise to numerous articles from different scientific fields (epidemiology, virology, immunology, airflow physics …) without any effort to link these different insights. In this review, we aim to establish relationships between epidemiological data and the characteristics of the virus strain responsible for the epidemic wave concerned. We have carried out this study on the Wuhan, Alpha, Delta and Omicron strains allowing us to illustrate the evolution of the relationships we have highlighted according to these different viral strains. We addressed the following questions. 1) How can the mean infectious dose (one quantum, by definition in epidemiology) be measured and expressed as an amount of viral RNA molecules (in genome units, GU) or as a number of replicative viral particles (in plaque-forming units, PFU)? 2) How many infectious quanta are exhaled by an infected person per unit of time? 3) How many infectious quanta are exhaled, on average, integrated over the whole contagious period? 4) How do these quantities relate to the epidemic reproduction rate R as measured in epidemiology, and to the viral load, as measured by molecular biological methods? 5) How has the infectious dose evolved with the different strains of SARS-CoV-2? We make use of state-of-the-art modelling, reviewed and explained in the appendix of the article (Supplemental Information, SI), to answer these questions using data from the literature in both epidemiology and virology. We have considered the modification of these relationships according to the vaccination status of the population.

Adaptations of intracellular bacteria to vacuolar or cytosolic niches.

Invasive bacteria colonise their host tissues by establishing niches inside eukaryotic cells, where they grow either in the cytosol or inside a specialised vacuole. These two distinct intracellular lifestyles both present benefits but also impose various constraints on pathogenic microorganisms, in terms of nutrient acquisition, space requirements, exposure to immune responses, and ability to disseminate. Here we review the major characteristics of cytosolic and vacuolar lifestyles and the strategies used by bacteria to overcome challenges specific to each compartment. Recent research providing evidence that these scenarios are not mutually exclusive is presented, with the dual lifestyles of two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, discussed in detail. Finally, we elaborate on the conceptual implications of polyvalence from the perspective of host-pathogen interactions.

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors.

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes.

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.

Engineering E. coli for Magnetic Control and the Spatial Localization of Functions.

The fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST.

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.

Exosome-mediated quality control: substrate recruitment and molecular activity.

The eukaryotic exosome is a multisubunit complex that is mainly responsible for 3'-5' exonucleolytic degradation of RNAs, both in the nucleus and the cytoplasm. In this review we summarize the recent experiments that have provided information on the organisation, structure and activity of this large assembly. Interestingly, eukaryotic exosomes have been implicated in a large number of RNA degradation pathways including recently discovered RNA quality control mechanisms. A variety of cofactors have been shown to participate in substrate recruitment and/or assist exonucleolytic activities. Despite this avalanche of new results, further analyses will be required to improve our understanding of exosome regulation.

An RNA-Binding Protein Secreted by a Bacterial Pathogen Modulates RIG-I Signaling.

RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-ß production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as "social RNAs" to trigger a host response during infection.

Sequential protein association with nascent 60S ribosomal particles.

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.