Hfq RIP-seq analysis
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ABSTRACT:
PROVIDER: PRJNA590055 | ENA |
REPOSITORIES: ENA
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Project description:In this study we used RNA co-immunoprecipitation followed by RNA-sequencing (RIP-seq) to identify Hfq-binding RNAs in Vibrio cholerae.
2020-02-03 | GSE140514 | GEO
Project description:Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of σ 70-dependent and independent promoters revealed that classical Escherichia coli-like σ70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
2017-03-21 | GSE85252 | GEO
Project description:This study represents the first attempt to characterize the RNA chaperone Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis.
2019-07-02 | PXD013953 | Pride
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.
2022-11-15 | GSE198671 | GEO
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.
2023-06-16 | GSE234792 | GEO
Project description:Bacterial small non-coding RNAs (sRNAs) play post-transcriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the P. aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5’ UTRs and sRNAs, and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that sRNAs association with Hfq is primarily a function of their expression levels, strongly supporting that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of post-transcriptional regulation in P. aeruginosa.
2019-11-15 | GSE136110 | GEO
Project description:Most bacterial small regulatory RNAs (sRNAs) modulate gene expression by forming complementary base pairs with target mRNAs, a process that is heavily reliant on the RNA chaperone Hfq in many microbes. Hfq, with its three distinct RNA-binding faces (proximal, rim, and distal), facilitates the simultaneous binding of sRNAs and target mRNAs, with each face providing specific interactions to promote pairing. However, the precise contributions of each Hfq face to RNA binding and sRNA-mRNA annealing in vivo remain unclear. Here, we systematically examined the functional impact of point mutations in chromomally-encoded Flag-tagged Hfq using the RNA Interaction by Ligation and Sequencing (RIL-seq) approach. While substantial numbers of sRNA-mRNA chimeras were detected for most Hfq face mutants, the rim face mutant R16A exhibited a near-complete loss of chimeras, even though functional assays confirmed that Hfq R16A retains partial regulatory activity. Further RIL-seq analysis demonstrated that the addition of the Flag tag did not significantly alter chimera formation or contribute to the loss of chimeras in R16A, and RNA immunoprecipitation sequencing (RIP-seq) analysis showed that R16A maintains partial RNA-binding activity. Using intracellular RIL-seq (iRIL-seq), a method with fewer in vitro processing steps after Hfq immunoprecipitation, we identified significantly more sRNA-mRNA chimeras in R16A compared to standard RIL-seq, indicating that the rim face plays a key role in stabilizing RNA pairs on Hfq. Our findings provide a comprehensive analysis of how the RNA-binding faces of Hfq contribute to sRNA stability and pairing in vivo and the unique function of the rim face. Additionally, we highlight the strengths of different RNA ligation-based sequencing approaches, with RIL-seq effectively capturing stable Hfq-associated interactions, while iRIL-seq captured more transient RNA pairings.
2025-12-17 | GSE296794 | GEO
Project description:Determination of the RNA interactome of the RNA-binding protein Hfq at three different time-point during growth on the three strains PAO1, PA14 and IHMA87, representing the three major P. aeruginosa phylogenetic lineages, using RIP-seq.
2022-02-07 | GSE171056 | GEO