Disturbance of the human gut microbiota in patients with Myotonic Dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a rare autosomal dominant genetic disorder. Although DM1 is primarily characterized by progressive muscular weakness, it exhibits many multisystemic manifestations, such as cognitive deficits, cardiac conduction abnormalities, and cataracts, as well as endocrine and reproductive issues. Additionally, the gastrointestinal (GI) tract is frequently affected, encompassing the entire digestive tract. However, the underlying causes of these GI symptoms remain uncertain, whether it is biomechanical problems of the intestine, involvement of bacterial communities, or both. The primary objective of this study is to investigate the structural changes in the gut microbiome of DM1 patients. To achieve this purpose, 35 patients with DM1 were recruited from the DM-Scope registry of the neuromuscular clinic in the Saguenay-Lac-St-Jean region of the province of Québec, Canada. Stool samples from these 35 patients, including 15 paired samples with family members living with them as controls, were collected. Subsequently, these samples were sequenced by 16S MiSeq and were analyzed with DADA2 to generate taxonomic signatures. Our analysis revealed that the DM1 status correlated with changes in gut bacterial community. Notably, there were differences in the relative abundance of Bacteroidota, Euryarchaeota, Fusobacteriota, and Cyanobacteria Phyla compared to healthy controls. However, no significant shift in gut microbiome community structure was observed between DM1 phenotypes. These findings provide valuable insights into how the gut bacterial community, in conjunction with biomechanical factors, could potentially influence the gastrointestinal tract of DM1 patients.

Optimizing Fecal Occult Blood Test (FOBT) Colorectal Cancer Screening Using Gut Bacteriome as a Biomarker.

BACKGROUND: Colorectal cancer (CRC) is a major cause of cancer mortality in the world. One of the most widely used screening tests for CRC is the immunochemical fecal occult blood test (iFOBT), which detects human hemoglobin from patient's stool sample. Although it is highly efficient in detecting blood from patients with gastro-intestinal lesions, such as polyps and cancers, the iFOBT has a high rate of false positive discovery. Recent studies suggested gut bacteria as a promising noninvasive biomarker for improving the diagnosis of CRC. In this study, we examined the composition of gut bacteria using iFOBT leftover from patients undergoing screening test along with a colonoscopy. METHODS: After collecting data from more than 800 patients, we considered 4 groups for this study. The first and second groups were respectively "healthy" in which the patients had either no blood in their stool or had blood but no lesions. The third and fourth groups of patients had both blood in their stools with precancerous and cancerous lesions and considered either as low-grade and high-grade lesion groups, respectively. An amplification of 16S rRNA (V4 region) gene was performed, followed by sequencing along with various statistical and bioinformatic analysis. RESULTS: We analyzed the composition of the gut bacteriome at phylum, class, genus, and species levels. Although members of the Firmicute phylum increased in the 3 groups compared to healthy patients, the phylum Actinobacteriota was found to decrease. Moreover, Blautia obeum and Anaerostipes hadrus from the phylum Firmicutes were increased and Collinsella aerofaciens from phylum Actinobacteriota was found decreased when healthy group is compared to the patients with high-grade lesions. Finally, among the 5 machine learning algorithms used to perform our analysis, both elastic net (AUC > 0.7) and random forest (AUC > 0.8) performs well in differentiating healthy patients from 3 other patient groups having blood in their stool. CONCLUSION: Our study integrates the iFOBT screening tool with gut bacterial composition to improve the prediction of CRC lesions.

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus.

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle-regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5' untranslated region (5' UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli.

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria.

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

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.

Immune System Modulations by Products of the Gut Microbiota.

The gut microbiota, which consists of all bacteria, viruses, fungus, and protozoa living in the intestine, and the immune system have co-evolved in a symbiotic relationship since the origin of the immune system. The bacterial community forming the microbiota plays an important role in the regulation of multiple aspects of the immune system. This regulation depends, among other things, on the production of a variety of metabolites by the microbiota. These metabolites range from small molecules to large macro-molecules. All types of immune cells from the host interact with these metabolites resulting in the activation of different pathways, which result in either positive or negative responses. The understanding of these pathways and their modulations will help establish the microbiota as a therapeutic target in the prevention and treatment of a variety of immune-related diseases.

GcvB small RNA uses two distinct seed regions to regulate an extensive targetome.

GcvB small RNA is described as post-transcriptional regulator of 1-2% of all mRNAs in Escherichia coli and Salmonella Typhimurium. At least 24 GcvB:mRNA interactions have been validated in vivo, establishing the largest characterized sRNA targetome. By performing MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we identified seven additional mRNAs negatively regulated by GcvB in E. coli. Contrary to the vast majority of previously known targets, which pair to the well-conserved GcvB R1 region, we validated four mRNAs targeted by GcvB R3 region. This indicates that base-pairing through R3 seed sequence seems relatively common. We also noticed unusual GcvB pairing sites in the coding sequence of two target mRNAs. One of these target mRNAs has a pairing site displaying a unique ACA motif, suggesting that GcvB could hijack a translational enhancer element. The second target mRNA is likely regulated via an active RNase E-mediated mRNA degradation mechanism. Remarkably, we confirmed the importance of the sRNA sponge SroC in the fine-tuning control of GcvB activity in function of growth conditions such as growth phase and nutrient availability.

Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli.

Small RNAs are key components of complex regulatory networks. These molecules can integrate multiple cellular signals to control specific target mRNAs. The recent development of high-throughput methods tremendously helped to characterize the full targetome of sRNAs. Using MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we reveal the targetomes of two sRNAs, CyaR and RprA. Interestingly, both CyaR and RprA interact with the 5'-UTR of hdeD mRNA, which encodes an acid-resistance membrane protein. We demonstrate that CyaR classically binds to the RBS of hdeD, interfering with translational initiation. We identified an A/U-rich motif on hdeD, which is bound by the RNA chaperone Hfq. Our results indicate that binding of this motif by Hfq is required for CyaR-induced degradation of hdeD mRNA. Additional data suggest that two molecules of RprA must bind the 5'-UTR of hdeD to block translation initiation. Surprisingly, while both CyaR and RprA sRNAs bind to the same motif on hdeD mRNA, RprA solely acts at the translational level, leaving the target RNA intact. By interchanging the seed region of CyaR and RprA sRNAs, we also swap their regulatory behavior. These results suggest that slight changes in the seed region could modulate the regulation of target mRNAs.

Translational control and Rho-dependent transcription termination are intimately linked in riboswitch regulation.

Riboswitches are regulatory elements that control gene expression by altering RNA structure upon the binding of specific metabolites. Although Bacillus subtilis riboswitches have been shown to control premature transcription termination, less is known about regulatory mechanisms employed by Escherichia coli riboswitches, which are predicted to regulate mostly at the level of translation initiation. Here, we present experimental evidence suggesting that the majority of known E. coli riboswitches control transcription termination by using the Rho transcription factor. In the case of the thiamin pyrophosphate-dependent thiM riboswitch, we find that Rho-dependent transcription termination is triggered as a consequence of translation repression. Using in vitro and in vivo assays, we show that the Rho-mediated regulation relies on RNA target elements located at the beginning of thiM coding region. Gene reporter assays indicate that relocating Rho target elements to a different gene induces transcription termination, demonstrating that such elements are modular domains controlling Rho. Our work provides strong evidence that translationally regulating riboswitches also regulate mRNA levels through an indirect control mechanism ensuring tight control of gene expression.

Identification of unknown RNA partners using MAPS.

Recent advances in high-throughput sequencing have led to an explosion in the rate of small regulatory RNAs (sRNAs) discovery among bacteria. However, only a handful of them are functionally characterized. Most of the time, little to no targets are known. In Lalaouna et al. (2015), we proposed a new technology to uncover sRNAs targetome, which is based on the MS2-affinity purification (MAPS). We were able to prove its efficiency by applying it on well-characterized sRNAs of Escherichia coli. Thereafter, we adapted the procedure to other kind of RNA (mRNAs and tRNA-derived RNA fragments) and bacteria (pathogenic or Gram-positive strains). Here, we clearly report all improvements and adjustments made to MAPS technology since it was originally reported.

Identification of bacterial sRNA regulatory targets using ribosome profiling.

Bacteria express large numbers of non-coding, regulatory RNAs known as 'small RNAs' (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets.

Small RNA-induced mRNA degradation achieved through both translation block and activated cleavage.

Small RNA (sRNA)-induced mRNA degradation occurs through binding of an sRNA to a target mRNA with the concomitant action of the RNA degradosome, which induces an endoribonuclease E (RNase E)-dependent cleavage and degradation of the targeted mRNA. Because many sRNAs bind at the ribosome-binding site (RBS), it is possible that the resulting translation block is sufficient to promote the rapid degradation of the targeted mRNA. Contrary to this mechanism, we report here that the pairing of the sRNA RyhB to the target mRNA sodB initiates mRNA degradation even in the absence of translation on the mRNA target. Remarkably, even though it pairs at the RBS, the sRNA RyhB induces mRNA cleavage in vivo at a distal site located >350 nucleotides (nt) downstream from the RBS, ruling out local cleavage near the pairing site. Both the RNA chaperone Hfq and the RNA degradosome are required for efficient cleavage at the distal site. Thus, beyond translation initiation block, sRNA-induced mRNA cleavage requires several unexpected steps, many of which are determined by structural features of the target mRNA.

Small RNA-induced differential degradation of the polycistronic mRNA iscRSUA.

Most polycistronic genes are expressed in a single transcript, in which each cistron produces a fixed amount of protein. In this report, we show the first example of differential degradation of a polycistronic gene induced by a small regulatory RNA (sRNA). Our data show that the iron-responsive sRNA, RyhB, binds to the second cistron of the polycistronic mRNA, iscRSUA, which encodes the necessary machinery for biosynthesis of Fe-S clusters, and promotes the cleavage of the downstream iscSUA transcript. This cleavage gives rise to the remaining 5'-section of the transcript encoding IscR, a transcriptional regulator responsible for activation and repression of several genes depending on the cellular Fe-S level. Our data indicate that the iscR transcript is stable and that translation is active. The stability of the iscR transcript depends on a 111-nucleotide long non-translated RNA section located between iscR and iscS, which forms a strong repetitive extragenic palindromic secondary structure and may protect against ribonucleases degradation. This novel regulation shows how sRNAs and mRNA structures can work together to modulate the transcriptional response to a specific stress.

The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis.

RyhB is a small RNA (sRNA) that downregulates about 20 genes involved in iron metabolism. It is expressed under low iron conditions and pairs with specific mRNAs to trigger their rapid degradation by the RNA degradosome. In contrast to this, another study has suggested that RyhB also activates several genes by increasing their mRNA level. Among these activated genes is shiA, which encodes a permease of shikimate, an aromatic compound participating in the biosynthesis of siderophores. Here, we demonstrate in vivo and in vitro that RyhB directly pairs at the 5'-untranslated region (5'-UTR) of the shiA mRNA to disrupt an intrinsic inhibitory structure that sequesters the ribosome-binding site (Shine-Dalgarno) and the first translation codon. This is the first demonstration of direct gene activation by RyhB, which has been exclusively described in degradation of mRNAs. Our physiological results indicate that the transported compound of the ShiA permease, shikimate, is important under conditions of RyhB expression, that is, iron starvation. This is demonstrated by growth assays in which shikimate or the siderophore enterochelin correct the growth defect observed for a ryhB mutant in iron-limited media.

RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli.

The small RNA RyhB has recently been shown to negatively regulate a number of mRNAs encoding dispensable iron-using proteins in Escherichia coli. The resulting decrease in the synthesis of iron-using proteins is thought to spare iron in order to ensure its availability for iron-requiring proteins that are indispensable. Indeed, the expression of RyhB from a heterologous promoter activates the iron-sensing repressor Fur, which suggests an increase in the pool of free intracellular iron (iron-sparing). In accordance with these observations, we report here that RyhB expression increases the concentration of free intracellular iron, as shown by direct measurements of the metal in whole cells by electron paramagnetic resonance spectroscopy. Our data also suggest that iron-sparing originates from rapid uptake of extracellular iron and not from already internalized metal. Furthermore, RyhB is shown to be essential for normal bacterial growth and survival during iron starvation, which is consistent with previous data describing the function of the small RNA. Overall, our data demonstrate that, by regulating synthesis of nonessential iron-using proteins, the small RNA RyhB ensures that the iron is directed towards the iron-requiring enzymes that are indispensable.