Hfq RIL-seq in Salmonella
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ABSTRACT:
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344
PROVIDER: GSE163336 | GEO | 2021/12/07
REPOSITORIES: GEO
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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.
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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).
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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.
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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.
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