Project description:We have developed a novel approach named LiRIP-seq to profile the global RNA-RNA interactome in Salmonella enterica. By pulse expressing T4 RNA ligase from an inducible pBAD promoter, LiRIP-seq enables in vivo proximity ligation of Hfq-bound RNAs to their interaction partners. This is followed by enrichment of ligation products (RNA chimeras) using Hfq-coIP and subsequent RNA-seq analysis.
Project description:In this study we used RIL-seq (RNA interaction by ligation and sequencing) to identify Hfq-mediated RNA-RNA interactions in V. cholerae.
Project description:A key challenge for understanding the role(s) played by short, non-coding RNAs (sRNAs) in bacteria is identifying the mRNA targets regulated by the sRNAs. Because the Hfq protein mediates the interactions between many sRNAs and the corresponding target mRNAs, one apporach to identify the mRNA targets of sRNAs is to capture sRNA:mRNA interactions occuring on Hfq by exposing cells to UV-irradiation, which forms cross-links between nucleic acids and proteins. We subjected cells of P. aeruginosa strain PAO1 and a derivative of PAO1 harboring a C-terminal VSV-G epitope on Hfq to UV-irradiation, immune-precipitated the Hfq-RNA complexes, ligated neighboring RNA molecules together with RNA Ligase, and then purififed the resulting RNAs. These RNAs were converted into cDNA libraries and sequenced using the Illumina NextSeq platform and then subjected to RIL-seq analysis pipeline (version 0.78) to identify chimeric RNA molecules. We also performed RNA-seq for PAO1 ∆phrS cells harboring an empty vector (pEV) or a vector expressing PhrS (pPhrS).
Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.
Project description:Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. The requirement of the RNA chaperone Hfq for the virulence of B. pertussis suggests that Hfq-dependent small regulatory RNAs (sRNAs) are involved in the virulence of this pathogen. To identify their potential mRNA targets, we applied a method combining experimental and computational approaches called RIL-seq. Our RIL-seq analysis revealed putative targets of several sRNAs including CT_532. This sRNA can interact with 5´UTR regions of mRNAs encoding the outer membrane proteins BP0840 and BP0943 (OmpA). The CT_532 sRNA shares 60% identity with the E. coli sRNA MicA and its expression is modulated by the heat and cold shocks as well as by osmotic stress. Collectively, these results suggest that CT_532 represents a MicA homolog. Importantly, the mutant lacking the first 22 nucleotides of CT_532 as well as its complemented variant overproducing CT_532 displayed reduced cytotoxicity towards human macrophages and impaired biofilm production compared to the wt strain. In addition, proteomic analysis revealed that CT_532 mutant produces increased amounts of OmpA protein.
Project description:RIL-seq experiment of EPEC hfq-flag mutant, in activating- conditions:growth on DMEM at 37°C to mid exponential growth phase (e.g., OD600=0.3). In these conditions EPEC strongly expresses its major virulence components, T3SS and BFP, mimicking infection. Non-activating conditions: overnight growth of static culture on LB medium at 37°C where virulence factors are not expressed. RIL-seq experiments are designed to reveal the interactions of sRNA and their targets.
Project description:In this study we take an integrative approach to capturing targets of the Hfq- associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica, using MS2 affinity purification and RNA- sequencing (MAPS).