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Polynucleotide phosphorylase can interact with small noncoding RNAs in both stabilizing and degradative modes

ABSTRACT: In all bacterial species examined thusfar, small regulatory RNAs (sRNAs) contribute to intricate patterns of genetic regulation. Many of the actions of these nucleic acids are mediated by chaperones such as the Hfq protein, and genetic screens have identified the exoribonuclease polynucleotide phosphorylase (PNPase) as a stabilizer and facilitator of sRNAs in vivo. We observe that the protective and facilitating effects of PNPase in vivo require the RNA-binding KH and S1 domains and the catalytic site, suggesting a requirement for physical interation of the ribonuclease with either the sRNA itself or other RNAs acting upstream of the process. Although purified PNPase can readily degrade sRNAs in vitro, those same substrates, as well as numerous other sRNAs and transcripts, can be co-purified from cells by immunoprecipitation with neither degradation nor modification to the 3’ end. Our results indicate that PNPase can bind RNA in two modes in vivo – either destructive or stabilizing, and that there is active flux of RNAs on PNPase so that the stable molecules do not accumulate. In the presence of Hfq, PNPase and sRNA form a ternary complex in which the enzyme plays a non-destructive, structural role, but the complex does not confer protection against the action of RNase E in vitro. Taken together, our results indicate that PNPase, Hfq and additional factors form a protective ribonucleoprotein assembly that stabilizes certain sRNAs and facilitates their activities.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE69856 | GEO | 2016/01/13

SECONDARY ACCESSION(S): PRJNA286969

REPOSITORIES: GEO

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Polynucleotide phosphorylase can interact with small noncoding RNAs in both stabilizing and degradative modes

Project description:In all bacterial species examined thusfar, small regulatory RNAs (sRNAs) contribute to intricate patterns of genetic regulation. Many of the actions of these nucleic acids are mediated by chaperones such as the Hfq protein, and genetic screens have identified the exoribonuclease polynucleotide phosphorylase (PNPase) as a stabilizer and facilitator of sRNAs in vivo. We observe that the protective and facilitating effects of PNPase in vivo require the RNA-binding KH and S1 domains and the catalytic site, suggesting a requirement for physical interation of the ribonuclease with either the sRNA itself or other RNAs acting upstream of the process. Although purified PNPase can readily degrade sRNAs in vitro, those same substrates, as well as numerous other sRNAs and transcripts, can be co-purified from cells by immunoprecipitation with neither degradation nor modification to the 3â end. Our results indicate that PNPase can bind RNA in two modes in vivo â either destructive or stabilizing, and that there is active flux of RNAs on PNPase so that the stable molecules do not accumulate. In the presence of Hfq, PNPase and sRNA form a ternary complex in which the enzyme plays a non-destructive, structural role, but the complex does not confer protection against the action of RNase E in vitro. Taken together, our results indicate that PNPase, Hfq and additional factors form a protective ribonucleoprotein assembly that stabilizes certain sRNAs and facilitates their activities. Using cells from the same original culture, immunoprecipitiations using the anti-FLAG M2 resin were performed in the presence or absence of tungstate. Total RNA and immunoprecipitated RNA were sequenced from two independent biological replicates. The number of normalized reads (RPKM) were compared between the total RNA (input) and the immunoprecipitated RNA (output).

2016-01-13 | E-GEOD-69856 | biostudies-arrayexpress

Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

Project description:In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay.

2019-07-08 | GSE125368 | GEO

RyhB-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 | GSE113591 | GEO

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

2021-09-22 | GSE113589 | GEO

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

2021-09-22 | GSE113587 | GEO

Transcriptome wide mapping of Hfq mediated RNA-RNA interactions in E. coli (RIL-seq)

Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.

2016-07-30 | E-MTAB-3910 | biostudies-arrayexpress

A novel acetyltransferase regulates the RNA binding capacity of the RNA chaperone Hfq in Escherichia coli

Project description:Bacterial small regulatory RNAs (sRNAs) regulate gene expression by base-pairing to their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, which binds sRNAs and mRNAs on different faces. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was identified as a novel silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and determined its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppresses all tested Hfq-dependent sRNA signaling. Chromosomal HqbA suppresses the signaling of the ChiX sRNA when the C-terminus of Hfq was deleted. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked interaction also interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, suggesting that HqbA is bifunctional, with separate roles for regulating via Hfq interaction and via acetylation of undefined substrates. Gel shift assays indicated that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs, but not high-affinity RNAs. Hfq-IP RNA-Seq in WT and chromosomal hqbA mutants led to the identification of two tRNA precursors, metZWV and proM, that were enriched in Hfq binding in the absence of HqbA interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

2023-11-13 | GSE235194 | 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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