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Identification of Hfq bound RNA by co-immunoprecipitation and RNA-sequencing

ABSTRACT: Co-immunoprecipitation with endogenous Hfq3xFlag in exponential, stationary, non-induced and virulence induced conditions, followed by RNA-sequencing, revealed 1697 mRNAs and 208 ncRNAs associated with Hfq. We identified 56 new ncRNAs and validated 3 Hfq-dependent trans sRNAs on by Northern blot analysis. Interestingly, 55% of the ncRNAs were encoded antisense to a protein coding sequence. Abundance of 4 asRNAs and their corresponding target mRNAs was altered upon hfq, indicating a substantial influence of Hfq on asRNA-target interactions.

ORGANISM(S): Agrobacterium tumefaciens

PROVIDER: GSE59123 | GEO | 2014/12/04

SECONDARY ACCESSION(S): PRJNA254473

REPOSITORIES: GEO

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Identification of Hfq bound RNA by co-immunoprecipitation and RNA-sequencing

Project description:Co-immunoprecipitation with endogenous Hfq3xFlag in exponential, stationary, non-induced and virulence induced conditions, followed by RNA-sequencing, revealed 1697 mRNAs and 208 ncRNAs associated with Hfq. We identified 56 new ncRNAs and validated 3 Hfq-dependent trans sRNAs on by Northern blot analysis. Interestingly, 55% of the ncRNAs were encoded antisense to a protein coding sequence. Abundance of 4 asRNAs and their corresponding target mRNAs was altered upon hfq, indicating a substantial influence of Hfq on asRNA-target interactions. 8 cDNA libraries were sequenced. A. tumefaciens hfq3xFlag and hfqWT (control) strains were grown to stationary and exponential growth phase and under non-induced and virulence induced conditions.

2014-12-04 | E-GEOD-59123 | biostudies-arrayexpress

Pervasive cis-antisense transcription negatively regulates gene expression in E. coli K12 in response to environmental changes

Project description:cis-antisense RNAs (cis-asRNAs) in prokaryotes are more pervasive than originally thought. However, little is known about their expression patterns and functions. Here we determined transcriptomes and proteomes of E. coli at multiple time points in five culture conditions using directional RNA-seq and tandem mass spectrometry. We found that a highly varying portion (27.2%~90.8%) of transcribed genes had cis-asRNAs dependent on growth phases and culture conditions, and that almost all transcribed genes changed their cis-asRNA levels relative to the mRNA level at different growth phases and culture conditions. Intriguingly, the correlation between the protein and mRNA levels of genes is abolished the increasing cis-asRNA levels relative to the mRNA levels, suggesting that cis-asRNAs might directly or indirectly inhibit translation by forming duplexes with mRNAs. All data sets are available for data mining from a unified resource to support further biological discoveries and insights into cis-asRNA-mediated gene expressional regulation.

2022-02-28 | PXD001640 | Pride

RNA-sequencing data from Pseudomonas putida KT2440 wild-type and Hfq-tagged strain form co-immunoprecipitation experiment

Project description:The RNA-chaperone Hfq is a global post-transcriptional regulator in Bacteria, important especially for the fine-tuning of the gene expression when the environmental conditions change. We looked into sRNAome during the growth of Pseudomonas putida KT2440 and compared it to a strain without Hfq protein. Numerous sRNAs were upregulated at a specific point during the growth and many were affected in the Hfq absence. Further we identified Hfq-binding RNA transcripts with co-immunoprecipitation and experimentally validated 17 sRNAs. Many novel Hfq-binding sRNAs were identified and are directly or indirectly regulated by Hfq. In addition 1870 mRNAs were found to bind to Hfq during the growth. Together, 202 antisense and 355 intergenic sRNAs have been detected in this study, several of them being 3’UTR- or 5’UTR-derived, and IS-related.

2017-01-01 | GSE85581 | GEO

Bacterial antisense RNAs are mainly the product of transcriptional noise

Project description:Cis-encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of the vast majority of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content, and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing 9 different asRNAs in M. pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

2016-01-18 | E-MTAB-4081 | biostudies-arrayexpress

Antisense RNA-mediated photosynthetic response in Synechocystis sp. PCC6803

Project description:Antisense RNAs (asRNAs) have diverse functions across three superkingdoms of life. However, their physiological roles for photosynthesis, the most efficient conversion system of solar energy and carbon dioxide into desirable biofuel, are elusive. To understand asRNA-mediated photosynthetic response, we systematically identified non-coding asRNAs and analyzed their differential regulation upon high light and/or low temperature. We found that large fractions of antisense regions are pervasively transcribed and differentially induced upon the change of light and/or temperature. Particularly, photosynthesis and ribosome related genes are mostly regulated by asRNA. Futhermore, we found that 93 long non-coding asRNAs spanning more than half of the cognate open reading frames (ORFs), unexpectedly. Intriguingly, many of them are associated with photosynthetic genes and they have positive role to the expression level of their cognate ORFs. Thus, our systematic transcriptome analysis of photosynthetic response indicates that asRNAs may finetune transcriptional response to enable efficient photosynthetic energy conversion.

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Bacterial antisense RNAs are mainly the product of transcriptional noise

2018-10-25 | PXD003231 | Pride

RNA co-immunoprecipitation with 3xFLAG::Hfq in Caulobacter crescentus

Project description:In this study we used RNA co-immunoprecipitation followed by RNA-sequencing to identify Hfq-binding RNAs in C. crescentus.

2022-12-31 | GSE148206 | GEO

Hfq Globally Binds and Destabilizes sRNAs and mRNAs in Yersinia pestis

Project description:Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3′ regions of mRNAs. An Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq-binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5′P-activated RNase E degradation pathway, which is consistent with a model in which Hfq facilitates sRNA-mRNA base pairing and the coupled degradation in Y. pestis. These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis.

2019-06-19 | GSE77555 | GEO

Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RNA-Seq]

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 | GSE136111 | GEO

Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RIP-Seq]

2019-11-15 | GSE136110 | GEO

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