RybB-mRNA pairing in absence of the RNA chaperone Hfq in vivo
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ABSTRACT:
PROVIDER: PRJNA453157 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7059132_1.fastq.gz | Fastqsanger.gz | |||
| SRR7059132_2.fastq.gz | Fastqsanger.gz | |||
| SRR7059133_1.fastq.gz | Fastqsanger.gz | |||
| SRR7059133_2.fastq.gz | Fastqsanger.gz | |||
| SRR7059134_1.fastq.gz | Fastqsanger.gz |
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Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.
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Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.
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Project description:sRNA-mRNA pairing in absence of the RNA chaperone Hfq
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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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