Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli.

Traffic of molecules across the bacterial membrane mainly relies on porins and transporters, whose expression must adapt to environmental conditions. To ensure bacterial fitness, synthesis and assembly of functional porins and transporters are regulated through a plethora of mechanisms. Among them, small regulatory RNAs (sRNAs) are known to be powerful post-transcriptional regulators. In Escherichia coli, the MicF sRNA is known to regulate only four targets, a very narrow targetome for a sRNA responding to various stresses, such as membrane stress, osmotic shock, or thermal shock. Using an in vivo pull-down assay combined with high-throughput RNA sequencing, we sought to identify new targets of MicF to better understand its role in the maintenance of cellular homoeostasis. Here, we report the first positively regulated target of MicF, the oppA mRNA. The OppA protein is the periplasmic component of the Opp ATP-binding cassette (ABC) oligopeptide transporter and regulates the import of short peptides, some of them bactericides. Mechanistic studies suggest that oppA translation is activated by MicF through a mechanism of action involving facilitated access to a translation-enhancing region in oppA 5'UTR. Intriguingly, MicF activation of oppA translation depends on cross-regulation by negative trans-acting effectors, the GcvB sRNA and the RNA chaperone protein Hfq.

Amygdala-driven apnea: A breath of fresh air in respiratory neurobiology.

The amygdala is mainly known for its role in the pathogenesis of anxiety and the initiation of fear responses. However, there is growing evidence showing that the amygdala's ability to respond to internal stimuli such as CO2 is limited, thereby challenging its role in the brain-behavior relationship. Based on these results and the strong inhibitory connections between the central nucleus of the amygdala and key brainstem areas regulating the reflexive respiratory responses to CO2, Feinstein et al. propose amygdala-driven apnea as a novel mechanism in the chemoreceptive origin of anxiety.

Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells.

RNA-binding proteins play myriad roles in regulating RNAs and RNA-mediated functions. In bacteria, the RNA chaperone Hfq is an important post-transcriptional gene regulator. Using live-cell super-resolution imaging, we can distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA-binding activity, with the distal face of Hfq contributing mostly to the mRNA binding in vivo. In addition, sRNAs can either co-occupy Hfq with the mRNA as a ternary complex, or displace the mRNA from Hfq in a binding face-dependent manner, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Finally, our data suggest that binding of Hfq to certain mRNAs through its distal face can recruit RNase E to promote turnover of these mRNAs in a sRNA-independent manner, and such regulatory function of Hfq can be decoyed by sRNA competitors that bind strongly at the distal face.

Messenger RNAs or mRNAs are molecules that the cell uses to transfer the information stored in the cell's DNA so it can be used to make proteins. Bacteria can regulate their levels of mRNA molecules, and they can therefore control how many proteins are being made, by producing a different type of RNA called small regulatory RNAs or sRNAs. Each sRNA can bind to several specific mRNA targets, and lead to their degradation by an enzyme called RNase E. Certain bacterial RNA-binding proteins, such as Hfq, protect sRNAs from being degraded, and help them find their mRNA targets. Hfq is abundant in bacteria. It is critical for bacterial growth under harsh conditions and it is involved in the process through which pathogenic bacteria infect cells. However, it is outnumbered by the many different RNA molecules in the cell, which compete for binding to the protein. It is not clear how Hfq prioritizes the different RNAs, or how binding to Hfq alters RNA regulation. Park, Prévost et al. imaged live bacterial cells to see how Hfq binds to RNA strands of different sizes. The experiments revealed that, when bacteria are growing normally, Hfq is mainly bound to mRNA molecules, and it can recruit RNase E to speed up mRNA degradation without the need for sRNAs. Park, Prévost et al. also showed that sRNAs could bind to Hfq by either replacing the bound mRNA or co-binding alongside it. The sRNA molecules that strongly bind Hfq can compete against mRNA for binding, and thus slow down the degradation of certain mRNAs. Hfq could be a potential drug target for treating bacterial infections. Understanding how it interacts with other molecules in bacteria could provide help in the development of new therapeutics. These findings suggest that a designed RNA that binds strongly to Hfq could disrupt its regulatory roles in bacteria, killing them. This could be a feasible drug design opportunity to counter the emergence of antibiotic-resistant bacteria.

Keeping Up with RNA-Based Regulation in Bacteria: New Roles for RNA Binding Proteins.

RNA binding proteins (RBPs) are ubiquitously found in all kingdoms of life. They are involved in a plethora of regulatory events, ranging from direct regulation of gene expression to guiding modification of RNA molecules. As bacterial regulators, RBPs can act alone or in concert with RNA-based regulators, such as small regulatory RNAs (sRNAs), riboswitches, or clustered regularly interspaced short palindromic repeats (CRISPR) RNAs. Various functions of RBPs, whether dependent or not on an RNA regulator, have been described in the past. However, the past decade has been a fertile ground for the development of novel high-throughput methods. These methods acted as stepping-stones for the discovery of new functions of RBPs and helped in the understanding of the molecular mechanisms behind previously described regulatory events. Here, we present an overview of the recently identified roles of major bacterial RBPs from different model organisms. Moreover, the tight relationship between RBPs and RNA-based regulators will be explored.

The small regulatory RNA Lpr10 regulates the expression of RpoS in Legionella pneumophila.

Legionella pneumophila (Lp) is a waterborne bacterium able to infect human alveolar macrophages, causing Legionnaires' disease. Lp can survive for several months in water, while searching for host cells to grow in, such as ciliates and amoeba. In Lp, the sigma factor RpoS is essential for survival in water. A previous transcriptomic study showed that RpoS positively regulates the small regulatory RNA Lpr10. In the present study, deletion of lpr10 results in an increased survival of Lp in water. Microarray analysis and RT-qPCR revealed that Lpr10 negatively regulates the expression of RpoS in the postexponential phase. Electrophoretic mobility shift assay and in-line probing showed that Lpr10 binds to a region upstream of the previously identified transcription start sites (TSS) of rpoS. A third putative transcription start site was identified by primer extension analysis, upstream of the Lpr10 binding site. In addition, nlpD TSS produces a polycistronic mRNA including the downstream gene rpoS, indicating a fourth TSS for rpoS. Our results suggest that the transcripts from the third and fourth TSS are negatively regulated by the Lpr10 sRNA. Therefore, we propose that Lpr10 is involved in a negative regulatory feedback loop to maintain expression of RpoS to an optimal level.

Trans-Acting Effectors Versus RNA Cis-Elements: A Tightly Knit Regulatory Mesh.

Prokaryotic organisms often react instantly to environmental variations to ensure their survival. They can achieve this by rapidly and specifically modulating translation, the critical step of protein synthesis. The translation machinery responds to an array of cis-acting elements, located on the RNA transcript, which dictate the fate of mRNAs. These cis-encoded elements, such as RNA structures or sequence motifs, interact with a variety of regulators, among them small regulatory RNAs. These small regulatory RNAs (sRNAs) are especially effective at modulating translation initiation through their interaction with cis-encoded mRNA elements. Here, through selected examples of canonical and non-canonical regulatory events, we demonstrate the intimate connection between mRNA cis-encoded features and sRNA-dependent translation regulation. We also address how sRNA-based mechanistic studies can drive the discovery of new roles for cis-elements. Finally, we briefly overview the challenges of using translation regulation by synthetic regulators as a tool.

On the Prowl: An In Vivo Method to Identify RNA Partners of a sRNA.

Bacterial cells dispose of numerous strategies to regulate gene expression. Small regulatory RNAs (sRNA) are pervasive molecules that allow gene expression regulation with exquisite precision. These molecules can bind mRNAs and negatively or positively modify their stability and interfere with translation. However, many features of sRNAs render identification of new targets or RNA interacting partners increasingly complex. In this chapter, we present a detailed procedure of MAPS, an in vivo technique based on the copurification of any type of RNA bound to an MS2-tagged sRNA. By focusing on the interaction between two RNAs rather than the outcome of this interaction, MAPS has proven useful in identifying unprecedented sRNA-RNA interactions. Below, we describe how to prepare MAPS samples and how to analyze RNA sequencing data files to determine enrichment ratios of different RNAs in an experimental condition vs a control condition. MAPS can be applied to most sRNAs of Escherichia coli and Salmonella spp., and can be easily optimized to more distant bacterial species.

Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action.

The first report of trans-acting RNA-based regulation in bacterial cells dates back to 1984. Subsequent studies in diverse bacteria unraveled shared properties of trans-acting small regulatory RNAs, forming a clear definition of these molecules. These shared characteristics have been used extensively to identify new small RNAs (sRNAs) and their interactomes. Recently however, emerging technologies able to resolve RNA-RNA interactions have identified new types of regulatory RNAs. In this review, we present a broader definition of trans-acting sRNA regulators and discuss their newly discovered intrinsic characteristics.

Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification Coupled to RNA Sequencing.

Small regulatory RNAs (sRNAs) are ubiquitous regulatory molecules expressed in living cells. In prokaryotes, sRNAs usually bind to target mRNAs to either promote their degradation or interfere with translation initiation. Because a single sRNA can regulate a considerable number of target mRNAs, we seek to identify those targets rapidly and reliably. Here, we present a robust method based on the co-purification of target mRNAs bound to MS2-tagged sRNAs expressed in vivo. After purification of the tagged-sRNA, we use RNAseq to determine the identity of all RNA interacting partners and their enrichment level. We describe how to analyze the RNAseq data through the Galaxy Project Platform bioinformatics tools to identify new mRNA targets. This technique is applicable to most sRNAs of E. coli and Salmonella.

Cellular Homeostasis: A Small RNA at the Crossroads of Iron and Photosynthesis.

The cyanobacterium Synechocystis relies on iron to perform oxygenic photosynthesis. This makes Synechocystis particularly sensitive to iron starvation. A new study shows that the small RNA IsaR1 is a major effector of the iron-stress response, remodeling the photosynthetic apparatus.

A game of tag: MAPS catches up on RNA interactomes.

In the last few decades, small regulatory RNA (sRNA) molecules emerged as key regulators in every kingdom of life. Resolving the full targetome of sRNAs has however remained a challenge. To address this, we used an in vivo tagging MS2-affinity purification protocol coupled with RNA sequencing technology, namely MAPS, to assemble full bacterial small RNAs targetomes. The impressive potential of MAPS has been supported by a number of reports. Here, we concisely overview RNA-tagging history that preceded the development of the MAPS assay and expose the range of possible uses of this technology.

Every little piece counts: the many faces of tRNA transcripts.

For over half a century, tRNAs have been exclusively known as decoders of genomic information. However, recent reports evidenced that tRNA transcripts are also bearers of functional RNAs, which are able to execute various tasks through an array of mechanisms. Here, we succinctly review the diversity and functions of RNAs deriving from tRNA loci.

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome.

A 3' external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise.

During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. However, we report that the 3'ETS of the glyW-cysT-leuZ polycistronic tRNA precursor is highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs are shown to base pair with the same region in the 3'ETS of leuZ (3'ETS(leuZ)). Disrupting the pairing by mutating 3'ETS(leuZ) strongly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3'ETS(leuZ) prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and decreases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3'ETS(leuZ) functions as a sponge to absorb transcriptional noise from repressed sRNAs. Additional data showing RybB and MicF sRNAs are co-purified with ITS(metZ-metW) and ITS(metW-metV) strongly suggest a wide distribution of this phenomenon.