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Identification of sRNA binding to ITSmetZW and ITSmetWV using MS2-affinity purification coupled with RNA sequencing (MAPS) technology

ABSTRACT: During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. The 3’ETS of the glyW-cysT-leuZ polycistronic tRNA precursor was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in the 3’ETS of leuZ (3’ETSleuZ). Disrupting the pairing by mutating 3’ETSleuZ significantly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3’ETSleuZ prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and increases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3’ETSleuZ functions as a sponge to absorb transcriptional noise from repressed sRNAs. Finally, the fact that RybB and MicF sRNAs are co-purified with ITSmetZ-metW and ITSmetW-metV strongly suggests a much broader phenomenon.

ORGANISM(S): Escherichia coli K-12

PROVIDER: GSE66517 | GEO | 2015/04/16

SECONDARY ACCESSION(S): PRJNA277198

REPOSITORIES: GEO

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Identification of sRNA binding to ITSmetZW and ITSmetWV using MS2-affinity purification coupled with RNA sequencing (MAPS) technology

Project description:During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. The 3’ETS of the glyW-cysT-leuZ polycistronic tRNA precursor was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in the 3’ETS of leuZ (3’ETSleuZ). Disrupting the pairing by mutating 3’ETSleuZ significantly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3’ETSleuZ prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and increases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3’ETSleuZ functions as a sponge to absorb transcriptional noise from repressed sRNAs. Finally, the fact that RybB and MicF sRNAs are co-purified with ITSmetZ-metW and ITSmetW-metV strongly suggests a much broader phenomenon. Identification of sRNAs co-purified with MS2-ITSmetZW and MS2-ITSmetWV. ITSmetZW and ITSmetWV (without MS2) were used as control

2015-04-16 | E-GEOD-66517 | biostudies-arrayexpress

Identification of RNA interacting with the sRNA sponge 3'ETSleuZ

Project description:During glyW-cysT-leuZ polycistronic tRNA maturation, the 3’external transcribed spacer (3’ETS) sequence is excised and act as a sRNA sponge (Lalaouna et al., 2015). Using MS2-affinity purification coupled with RNA sequencing (MAPS), we demonstrated that 3’ETSleuZ was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in 3’ETSleuZ. Here, we use MS2-3’ETSleuZ as bait to co-purify all interacting sRNAs and confirm 3’ETSleuZ/RyhB and 3’ETSleuZ/RybB interactions.

2017-11-28 | GSE79278 | GEO

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RprA sRNA targetome.

Project description:Recently, we developed an in vivo technology to draw the interacting map of a specific small regulatory RNA (sRNA). We called it MAPS for MS2-affinity purification coupled with RNA sequencing. Using this technology, we already revealed the targetome of RyhB, RybB and DsrA, three well-characterized sRNAs in Escherichia coli. In this study, we perform MAPS with RprA sRNA.

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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals GcvB sRNA targetome.

2018-12-06 | GSE80019 | GEO

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals CyaR sRNA targetome in Escherichia coli.

2018-01-03 | GSE90128 | GEO

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RybB sRNA targetome

Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RybB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RybB in a rne131 ΔrybB strain. RybB (without MS2) was used as control

2015-04-16 | E-GEOD-66518 | biostudies-arrayexpress

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RyhB sRNA targetome

Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RyhB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RyhB in a rne131 ?ryhB strain. RyhB (without MS2) was used as control

2015-04-16 | E-GEOD-66519 | biostudies-arrayexpress

RybB-mRNA pairing in absence of the RNA chaperone Hfq in vivo

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.

2021-09-22 | GSE113589 | GEO

DsrA-mRNA pairing in absence of the RNA chaperone Hfq in vivo

2021-09-22 | GSE113587 | GEO

RyhB-mRNA pairing in absence of the RNA chaperone Hfq in vivo

2021-09-22 | GSE113591 | GEO