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

ABSTRACT: 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

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Eric Massé

PROVIDER: E-GEOD-66518 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

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

MS2-affinity purification coupled with RNAseq (MAPS) revealed functional tRNA-derived RNA fragments

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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Affinity chromatography-based identification of sRNA protein partners in Rhizobia

Project description:This project aims to identify novel protein partners of conserved trans-sRNAs in the α-proteobacterium Sinorhizobium meliloti. Trans-sRNAs (AbcR2, NfeR1 and EcpR1) were tagged at their 5’ ends with the MS2 aptamer. To identify the proteins associated with the trans-sRNAs we: i) expressed and purified the MS2 coat protein fused to maltose-binding protein (MS2-MBP); ii) purified MS2 tagged sRNAs conjugated with MS2-MBP via amylase column; and iii) subjected retained proteins to mass spectrometry analysis. To discard unspecific interactions we also analysed several control samples: i) samples expressing untagged trans-sRNAs; ii) samples expressing an MS2 tagged Rho-independent transcriptional terminator.

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Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure

Project description:Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than experimentally detected under given conditions. This raises the question whether the sRNA target specificity can change upon metabolic or environmental changes. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator, which is located upstream of the Trp biosynthesis gene trpE(G) and harbors the small ORF trpL. When Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which then downregulates the trpDC operon. On the other hand, the trpL-encoded leader peptide peTrpL has a Trp-independent role in posttranscriptional regulation of antibiotic resistance. Here, we show that upon tetracycline (Tc) exposure, rnTrpL accumulates independently of Trp availability. Further, we provide evidence that both rnTrpL and peTrpL act together in the Tc-dependent destabilization of rplUrpmA mRNA encoding ribosomal proteins L21 and L27. rnTrpL, peTrpL and rplUrpmA mRNA were copurified in an antibiotic- dependent ribonucleoprotein complex (ARNP). In vitro ARNP reconstitution with competing trpD and rplU transcripts revealed that peTrpL and Tc reprogram the rnTrpL specificity in favor of rplU. In vivo, this probably supports bacterial adaptation to antibiotics. Our findings provide evidence for sRNA reprograming in response to antibiotic exposure.

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

2015-04-16 | GSE66519 | GEO

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

2015-04-16 | GSE66518 | GEO

psiCLIP of SmB, Prp16 and Prp22 in C, C* and P spliceosomal complexes

Project description:Despite the major progress made into spliceosome mechanisms through CryoEM, a major obstacle of this approach is its inability to map the positioning of helicases on the RNA substrate. Here we present a method ‘psiCLIP’ which probes protein-RNA interactions in specific spliceosomal states. The method is based on iCLIP, but is different to previous iCLIP studies in that it is performed on step-specific spliceosomes that are prepared from in vitro splicing extracts, similarly to those prepared for CryoEM. We applied psiCLIP to SmB (C complex), Prp16 (C complex) and Prp22 (C* and P complexes), using pre-mRNA substrates based on UBC4 and ACT1. We also used ATPase deficient dominant negative (dn) mutants of Prp16 and Prp22 to determine the contribution of ATPase activity to binding patterns.

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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaG sRNA targetome

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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

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