Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in Bacteria.

In this chapter, we describe in detail how to perform a successful smFISH experiment and how to quantify mRNA transcripts in bacterial cells. The flexibility of the method allows for straightforward adaptation to different bacterial species and experimental conditions. Thanks to the feasibility of the approach, the method can easily be adapted by other laboratories. Finally, we believe that this method has a great potential to generate insights into the complicated life of bacteria.

Physiological stress drives the emergence of a Salmonella subpopulation through ribosomal RNA regulation.

Bacteria undergo cycles of growth and starvation to which they must adapt swiftly. One important strategy for adjusting growth rates relies on ribosomal levels. Although high ribosomal levels are required for fast growth, their dynamics during starvation remain unclear. Here, we analyzed ribosomal RNA (rRNA) content of individual Salmonella cells by using fluorescence in situ hybridization (rRNA-FISH) and measured a dramatic decrease in rRNA numbers only in a subpopulation during nutrient limitation, resulting in a bimodal distribution of cells with high and low rRNA content. During nutritional upshifts, the two subpopulations were associated with distinct phenotypes. Using a transposon screen coupled with rRNA-FISH, we identified two mutants, DksA and RNase I, acting on rRNA transcription shutdown and degradation, which abolished the formation of the subpopulation with low rRNA content. Our work identifies a bacterial mechanism for regulation of ribosomal bimodality that may be beneficial for population survival during starvation.

Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks.

Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA sequencing (scRNA-seq) is increasingly used to study the host factors underlying diverse cellular phenotypes but has limited capacity to analyze the role of bacterial factors. Here, we developed scPAIR-seq, a single-cell approach to analyze infection with a pooled library of multiplex-tagged, barcoded bacterial mutants. Infected host cells and barcodes of intracellular bacterial mutants are both captured by scRNA-seq to functionally analyze mutant-dependent changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We analyzed redundancy between effectors and mutant-specific unique fingerprints and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

Alternative 3' UTRs direct localization of functionally diverse protein isoforms in neuronal compartments.

The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs-alternative splicing and polyadenylation-have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3'UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

RNA localization is a key determinant of neurite-enriched proteome.

Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity.Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity.

Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution.

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.