Small Non-Coding RNAs and Their Role in Locoregional Metastasis and Outcomes in Early-Stage Breast Cancer Patients.

Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.

SARS-CoV-2 remodels the landscape of small non-coding RNAs with infection time and symptom severity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides. Host-derived sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and small nucleolar RNA-derived small RNAs (sdRNAs) exhibited significant differential expression in infected patients compared to controls. Importantly, miRNA expression was predominantly down-regulated in response to SARS-CoV-2 infection, especially in patients with severe symptoms. Furthermore, we identified specific tsRNAs derived from Glu- and Gly-tRNAs as major altered elements upon infection, with 5' tRNA halves being the most abundant species and suggesting their potential as biomarkers for viral presence and disease severity prediction. Additionally, down-regulation of C/D-box sdRNAs and altered expression of tinyRNAs (tyRNAs) were observed in infected patients. These findings provide valuable insights into the host sncRNA response to SARS-CoV-2 infection and may contribute to the development of further diagnostic and therapeutic strategies in the clinic.

Development of synthetic small regulatory RNA for Rhodococcus erythropolis.

Rhodococci have been regarded as ideal chassis for biotransformation, biodegradation, and biosynthesis for their unique environmental persistence and robustness. However, most species of Rhodococcus are still difficult to metabolically engineer due to the lack of genetic tools and techniques. In this study, synthetic sRNA strategy was exploited for gene repression in R. erythropolis XP. The synthetic sRNA based on the RhlS scaffold from Pseudomonas aeruginosa functions better in repressing sfgfp expression than those based on E. coli MicC, SgrS, and P. aeruginosa PrrF1-2 scaffold. The RhlS-based sRNAs were applied to study the influence of sulfur metabolism on biodesulfurization (BDS) efficiency in R. erythropolis XP and successfully identified two genes involved in sulfur metabolism that affect the BDS efficiency significantly. The RhlS-based synthetic sRNAs show promise in the metabolic engineering of Rhodococcus and promote the industrial applications of Rhodococcus in environmental remediation and biosynthesis.

In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria.

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described.

Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σE-dependent small RNA with unusual mRNA activation activity.

The oral commensal Fusobacterium nucleatum can spread to extra-oral sites, where it is associated with diverse pathologies, including pre-term birth and cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of the RNA regulatory networks that allow this bacterium to adapt to its various niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor, σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped the 5' and 3' ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156-nucleotide sRNA previously misannotated as the 5' untranslated region (UTR) of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as is FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5' and 3' UTRs in F. nucleatum and expands our understanding of the σE response in this evolutionarily distant bacterium.IMPORTANCEThe oral microbe Fusobacterium nucleatum can colonize secondary sites, including cancer tissue, and likely deploys complex regulatory systems to adapt to these new environments. These systems are largely unknown, partly due to the phylogenetic distance of F. nucleatum to other model organisms. Previously, we identified a global stress response mediated by σE that displays functional conservation with the envelope stress response in Proteobacteria, comprising a coding and noncoding regulatory arm. Through global identification of transcriptional start and stop sites, we uncovered the small RNA (sRNA) FoxJ as a novel component of the noncoding arm of the σE response in F. nucleatum. Together with its companion sRNA FoxI, FoxJ post-transcriptionally modulates the synthesis of envelope proteins, revealing a conserved function for σE-dependent sRNAs between Fusobacteriota and Proteobacteria. Moreover, FoxJ activates the gene expression for several targets, which is a mode of regulation previously unseen in the noncoding arm of the σE response.

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.

Agnodice: indexing experimentally supported bacterial sRNA-RNA interactions.

In the last decade, the immense growth in the field of bacterial small RNAs (sRNAs), along with the biotechnological breakthroughs in Deep Sequencing permitted the deeper understanding of sRNA-RNA interactions. However, microbiology is currently lacking a thoroughly curated collection of this rapidly expanding universe. We present Agnodice (https://dianalab.e-ce.uth.gr/agnodice), our effort to systematically catalog and annotate experimentally supported bacterial sRNA-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Deep Sequencing interactome identification techniques. It comprises 39,600 entries which are annotated at strain-level resolution and pertain to 399 sRNAs and 12,137 target RNAs identified in 71 bacterial strains. The database content is exclusively experimentally supported, incorporating interactions derived via low yield as well as state-of-the-art high-throughput methods. The entire content of the database is freely accessible and can be directly downloaded for further analysis. Agnodice will serve as a valuable source, enabling microbiologists to form novel hypotheses, design/identify novel sRNA-based drug targets, and explore the therapeutic potential of microbiomes from the perspective of small regulatory RNAs.IMPORTANCEAgnodice (https://dianalab.e-ce.uth.gr/agnodice) is an effort to systematically catalog and annotate experimentally supported bacterial small RNA (sRNA)-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Next Generation Sequencing interactome identification techniques.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as "functional amyloids". In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

The serum small non-coding RNA (SncRNA) landscape as a molecular biomarker of age associated muscle dysregulation and insulin resistance in older adults.

Small noncoding RNAs (sncRNAs) are implicated in age-associated pathologies, including sarcopenia and insulin resistance (IR). As potential circulating biomarkers, most studies have focussed on microRNAs (miRNAs), one class of sncRNA. This study characterized the wider circulating sncRNA transcriptome of older individuals and associations with sarcopenia and IR. sncRNA expression including miRNAs, transfer RNAs (tRNAs), tRNA-associated fragments (tRFs), and piwi-interacting RNAs (piRNAs) was measured in serum from 21 healthy and 21 sarcopenic Hertfordshire Sarcopenia Study extension women matched for age (mean 78.9 years) and HOMA2-IR. Associations with age, sarcopenia and HOMA2-IR were examined and predicted gene targets and biological pathways characterized. Of the total sncRNA among healthy controls, piRNAs were most abundant (85.3%), followed by tRNAs (4.1%), miRNAs (2.7%), and tRFs (0.5%). Age was associated (FDR < 0.05) with 2 miRNAs, 58 tRNAs, and 14 tRFs, with chromatin organization, WNT signaling, and response to stress enriched among gene targets. Sarcopenia was nominally associated (p < .05) with 12 tRNAs, 3 tRFs, and 6 piRNAs, with target genes linked to cell proliferation and differentiation such as Notch Receptor 1 (NOTCH1), DISC1 scaffold protein (DISC1), and GLI family zinc finger-2 (GLI2). HOMA2-IR was nominally associated (p<0.05) with 6 miRNAs, 9 tRNAs, 1 tRF, and 19 piRNAs, linked with lysine degradation, circadian rhythm, and fatty acid biosynthesis pathways. These findings identify changes in circulating sncRNA expression in human serum associated with chronological age, sarcopenia, and IR. These may have clinical utility as circulating biomarkers of ageing and age-associated pathologies and provide novel targets for therapeutic intervention.

A landscape of mouse mitochondrial small non-coding RNAs.

Small non-coding RNAs (ncRNAs), particularly miRNAs, play key roles in a plethora of biological processes both in health and disease. Although largely operative in the cytoplasm, emerging data indicate their shuttling in different subcellular compartments. Given the central role of mitochondria in cellular homeostasis, here we systematically profiled their small ncRNAs content across mouse tissues that largely rely on mitochondria functioning. The ubiquitous presence of piRNAs in mitochondria (mitopiRNA) of somatic tissues is reported for the first time, supporting the idea of a strong and general connection between mitochondria biology and piRNA pathways. Then, we found groups of tissue-shared and tissue-specific mitochondrial miRNAs (mitomiRs), potentially related to the "basic" or "cell context dependent" biology of mitochondria. Overall, this large data platform will be useful to deepen the knowledge about small ncRNAs processing and their governed regulatory networks contributing to mitochondria functions.

Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells.

BACKGROUND: Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution. METHODS: Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas. RESULTS: Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area. CONCLUSIONS: This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron.

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

Evaluation of 5'-End Phosphorylation for Small RNA Stability and Target Regulation In Vivo.

Bacterial small RNAs (sRNAs) can be equipped at the 5' end with triphosphate (5'PPP) or monophosphate (5'P) groups, depending on whether they are primary transcripts, undergo dephosphorylation or originate via processing. Often, 5' groups hallmark RNAs for rapid decay, but whether this also applies to sRNAs is little explored. Moreover, the sRNA 5'P group could activate endoribonuclease RNase E to cleave the base-paired target RNA, but a tool for investigation in vivo was lacking. Here, we describe a two-plasmid system suitable for the generation of 5' monophosphorylated RNAs on demand inside the cell. The sRNA gene of interest is fused to the 3' end of a fragment of sRNA GlmZ and transcribed from a plasmid in an IPTG-inducible manner. The fusion RNA gets cleaved upon arabinose-controlled expression of rapZ, provided on a compatible plasmid. Adaptor protein RapZ binds the GlmZ aptamer and directs RNase E to release the sRNA of choice with 5'P ends. An isogenic plasmid generating the same sRNA with a 5'PPP end allows for direct comparison. The fates of the sRNA variants and target RNA(s) are monitored by Northern blotting. This tool is applicable to E. coli and likely other enteric bacteria.

Discovering Novel Bacterial Small RNA by RNA-seq Analysis Toolkit ANNOgesic.

ANNOgesic is an RNA-seq analysis pipeline that can detect sRNAs and many other genomic features in bacteria and archaea. In addition to listing sRNA candidates, ANNOgesic also generates various formats of data files for visual examination and downstream experimental design. Based on validations from previous studies, the sRNA predictions are accurate and reliable. In this chapter, we outline the sRNA detection algorithm, important parameters used, step-by-step execution, and data interpretation with a B. pertussis study as an example. Following those procedures, novel sRNA can be revealed by ANNOgesic.

CRISPR Interference-Based Functional Small RNA Genomics.

Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at-or migration along-predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.

Analysis of Phage Regulatory RNAs: Sequencing Library Construction from the Fraction of Small Prokaryotic RNAs Less Than 50 Nucleotides in Length.

So far, bacterial regulatory sRNAs of length less than 50 nucleotides have been poorly understood, and a low number of such molecules has been identified. The first microRNA-size functional ribonucleic acid occurring in a bacterial cell has been described only recently, and it was found to be encoded by a bacteriophage. One of the reasons for such a scarcity in this field is the lack of procedures intended for the isolation and selection of molecules of this size from bacterial cells. To meet these difficulties, we describe here the few-step procedure of isolation, purification, selection, and sequencing library preparation that is dedicated to the fraction of very small, bacterial RNA molecules.

Defining Bacterial RNA-RNA Interactomes Using CLASH.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen accounting for high mortality rates among infected patients. Transcriptomic regulation by small RNAs (sRNAs) has been shown to regulate networks promoting antibiotic resistance and virulence in S. aureus. Yet, the biological role of most sRNAs during MRSA host infection remains unknown. To fill this gap, in collaboration with the lab of Jai Tree, we performed comprehensive RNA-RNA interactome analyses in MRSA using CLASH under conditions that mimic the host environment. Here we present a detailed version of this optimized CLASH (cross-linking, ligation, and sequencing of hybrids) protocol we recently developed, which has been tailored to explore the RNA interactome in S. aureus as well as other Gram-positive bacteria. Alongside, we introduce a compilation of helpful Python functions for analyzing folding energies of putative RNA-RNA interactions and streamlining sRNA and mRNA seed discovery in CLASH data. In the accompanying computational demonstration, we aim to establish a standardized strategy to evaluate the likelihood that observed chimeras arise from true RNA-RNA interactions.