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Hfq‐binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base‐pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non‐targets among the mRNAs predicted to base pair with Hfq‐binding sRNAs is poorly understood. Using the Hfq‐binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base‐pairing in single or multiple base‐pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non‐targets predicted to base pair with Spot 42 lacked an Hfq‐binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq‐binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non‐target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs. Target recognition by bacterial small RNAs is not well understood. The accessibility of the targeted sequences, stability of the duplex, and the binding and positioning of the RNA chaperone Hfq on the mRNA are critical.

▪ Abstract  Small noncoding RNAs have been found in all organisms, primarily as regulators of translation and message stability. The most exhaustive searches have taken place in E. coli, resulting in identification of more than 50 small RNAs, or 1%–2% of the number of protein-coding genes. One large class of these small RNAs uses the RNA chaperone Hfq; members of this class act by pairing to target messenger RNAs. Among the members of this class are DsrA and RprA, which positively regulate rpoS translation, OxyS, which negatively regulates rpoS translation and fhlA translation, RyhB, which reapportions iron use in the cell by downregulating translation of many genes that encode Fe-containing proteins, and Spot 42, which changes the polarity of translation in the gal operon. The promoters of these small RNAs are tightly regulated, frequently as part of well-understood regulons. Lessons learned from the study of small RNAs in E. coli can be applied to finding these important regulators in other organisms.

Significance Most sRNAs of Escherichia coli function at the 5′ end of the target RNA. Binding of sRNA to the 5′ end of the target RNA induces a ribosome-free zone that causes molecular events such as target RNA degradation and Rho-termination. Results from this study show that Spot 42 enhances Rho-termination at the end of the galT gene, demonstrating for the first time that sRNA could function at the 3′ end of the target RNA. The region where Spot 42 binds overlaps with two other functional cis-acting sites: the ribosome-binding site for galK and the cytosine-rich, guanine-poor region known as the Rho-binding site, suggesting a unique molecular mechanism to enhance Rho-termination occurring at a cistron junction in a multicistronic operon. The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM . During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM . However, during late-log growth, type 1 polarity is established in which galK is greater than galT , as follows: galE > galK > galT > galM . We found that type 2 polarity occurs as a result of the down-regulation of galK , which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK- specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT . Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT .

The cytotoxicity of Vibrio parahaemolyticus has been related to the type III secretion system 1 effector protein VP1680, which is secreted and translocated into host cells with the help of the specific chaperone protein, VP1682. This study sought to confirm the in silico analysis, which predicted that a small regulatory RNA (Spot 42) could base pair with the region encompassing the ribosomal-binding site and initiation codon of the vp1682 mRNA. Electrophoresis mobility shift assays indicated that Spot 42 could bind to the vp1682 mRNA with the help of Hfq. Consistent with these results, the translation of the vp1682 mRNA was inhibited when both Hfq and Spot 42 were added to the in vitro translation reaction. The cytotoxic activity against infected Caco-2 cells was significantly increased in the Spot 42 deletion mutant (Δspf) at 4 h after infection as compared with the parental strain. Additionally, we observed that both VP1682 and VP1680 were more highly expressed in Δspf mutants than in the parental strain. These results indicate that Spot 42 post-transcriptionally regulates the expression of VP1682 in V. parahaemolyticus, which contributes to cytotoxicity in vivo. The Vibrio parahaemolyticus sRNA Spot 42 inhibits the expression of VP1682 by base pairing with the region encompassing the RBS and initiation codon of the vp1682 mRNA and thereby reduces cytotoxicity.

Bacterial regulatory small RNAs (sRNAs) have shown promise for gene knock‐down studies and metabolic engineering. However, some mRNAs might be difficult to target due to poor binding by the Hfq chaperone, individual synthetic sRNAs can have off‐target effects, potential sRNA toxicities have not been studied globally, and a consensus on optimal sRNA design has yet to emerge. Here, Spot 42 sRNA is validated as an excellent scaffold by showing that its over‐expression minimally affects the growth rate of Escherichia coli, and that inhibition is reliably achieved for all eight tested protein targets by designing antisense to target the first few codons. Two related sRNAs that could not be cloned, possibly due to lethality of the encoded sRNAs, became clonable when an eight‐nucleotide sequence was inserted directly upstream of the antisense region. Global fitness costs for E. coli of the designer sRNAs were measured and found to be variable but tolerable. Importantly for utility, there was no correlation between target inhibition and cellular toxicity. As a proof of concept for applications, suppression of the UAG stop codon was improved by knock down of translation release factor 1 (RF1). A plasmid‐encoded small RNA designed to hybridize with the first few codons of release factor 1 mRNA decreased the protein level which increased β‐galactosidase α‐peptide suppression which then turned E. coli yellow.

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2 ) occur in multiple types of cancer 1 . However, clinical responses to FGFR inhibitors have remained variable 1 – 9 , emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening 10 , 11 and tumour modelling in mice 12 , 13 , and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1–E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 ( FGFR2 ΔE18 ). Functional in vitro and in vivo examination of a compendium of FGFR2 ΔE18 and full-length variants pinpointed FGFR2 -E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2 ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies. Truncation of exon 18 of FGFR2 ( FGFR2 ΔE18 ) is a potent driver mutation in mice and humans, and FGFR-targeted therapy should be considered for patients with cancer expressing stable FGFR2 ΔE18 variants.

Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterize the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a 2-yr period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli . The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonized patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE. Modelling combined with epidemiological and genomic data analysis from a hospital in Spain reveals frequent transmission of high-risk pOXA-48-carrying enterobacteria clones between patients and horizontal transfer of pOXA-48 plasmid within patients.

The substrates of the Brazilian campos rupestre s, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres -dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae . We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae . We highlight that plant-associated microbial populations in the campos rupestre s harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

With Alzheimer’s disease (AD) exhibiting reduced ability of neural stem cell renewal, we hypothesized that de novo mutations controlling embryonic development, in the form of brain somatic mutations instigate the disease. A leading gene presenting heterozygous dominant de novo autism-intellectual disabilities (ID) causing mutations is activity-dependent neuroprotective protein (ADNP), with intact ADNP protecting against AD-tauopathy. We discovered a genomic autism ADNP mutation (c.2188C>T) in postmortem AD olfactory bulbs and hippocampi. RNA-Seq of olfactory bulbs also identified a novel ADNP hotspot mutation, c.2187_2188insA. Altogether, 665 mutations in 596 genes with 441 mutations in AD patients (389 genes, 38% AD—exclusive mutations) and 104 genes presenting disease-causing mutations (OMIM) were discovered. OMIM AD mutated genes converged on cytoskeletal mechanisms, autism and ID causing mutations (about 40% each). The number and average frequencies of AD-related mutations per subject were higher in AD subjects compared to controls. RNA-seq datamining (hippocampus, dorsolateral prefrontal cortex, fusiform gyrus and superior frontal gyrus—583 subjects) yielded similar results. Overlapping all tested brain areas identified unique and shared mutations, with ADNP singled out as a gene associated with autism/ID/AD and presenting several unique aging/AD mutations. The large fusiform gyrus library (117 subjects) with high sequencing coverage correlated the c.2187_2188insA ADNP mutation frequency to Braak stage (tauopathy) and showed more ADNP mutations in AD specimens. In cell cultures, the ADNP-derived snippet NAP inhibited mutated-ADNP-microtubule (MT) toxicity and enhanced Tau–MT association. We propose a paradigm-shifting concept in the perception of AD whereby accumulating mosaic somatic mutations promote brain pathology.

Inhibition of T cell infiltration dampens antitumor immunity and causes resistance to immune checkpoint blockade (ICB) therapy. By in vivo CRISPR screening in B16F10 melanoma in female mice, here we report that loss of melanocortin-1 receptor (MC1R) in melanoma cells activates antitumor T cell response and overcomes resistance to ICB. Depletion of MC1R from another melanocytic melanoma model HCmel1274 also enhances ICB efficacy. By activating the GNAS-PKA axis, MC1R inhibits interferon-gamma induced CXCL9 / 10 / 11 transcription, thus impairing T cell infiltration into the tumor microenvironment. In human melanomas, high MC1R expression correlates with reduced CXCL9 / 10 / 11 expression, impaired T cell infiltration, and poor patient prognosis. Whereas MC1R activation is restricted to melanoma, GNAS activation by hotspot mutations is observed across diverse cancer types and is associated with reduced CXCL9 / 10 / 11 expression. Our study implicates MC1R as a melanoma immunotherapy target and suggests GNAS-PKA signaling as a pan-cancer oncogenic pathway inhibiting antitumor T cell response. Aberrant G protein-coupled receptor (GPCR) signaling has been associated with tumor progression and metastasis. Here the authors show that depletion of the GPCR melanocortin-1 receptor (MC1R) in melanoma cells is associated with enhanced T cell infiltration and anti-tumor immune responses.