Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

Chihara, Kotaro; Bischler, Thorsten; Barquist, Lars; Monzon, Vivian; Noda, Naohiro; Vogel, Jörg; Tsuneda, Satoshi

doi:10.1128/msystems.00590-19

Cited by 22 publications

(30 citation statements)

References 70 publications

(121 reference statements)

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“…CLIP‐seq data analysis was performed as described previously (Chihara et al , ). Briefly, read 1 and read 2 files containing the paired‐end reads were quality and adapter trimmed via Cutadapt (Martin, ), version 1.17, using a cutoff Phred score of 20 in NextSeq mode, and reads without any remaining bases were discarded.…”

Section: Methodsmentioning

confidence: 99%

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

et al. 2020

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RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNAprotein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of~88% of the transcripts and~62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.

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Section: Methodsmentioning

confidence: 99%

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

et al. 2020

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“…We also predicted a total of 55 proteins to be synthesized from leaderless mRNAs (pTSS <10 bp of the start codon), which may require a distinct type of translation initiation [32]. P. aeruginosa is rife with sRNAs, many of which associate with the sRNA chaperone Hfq [33,34] and act by base-paring mechanisms to either repress or activate the translation of trans-encoded target mRNAs [35][36][37][38]. Although many more have been predicted [39], PseudoCAP [17] currently lists only 43 ncRNAs for this strain, most of which were expressed during late exponential/stationary phase [40].…”

Section: The Primary Transcriptome Of Uninfected P Aeruginosa Pao1mentioning

confidence: 99%

Introducing differential RNA-seq mapping to track the early infection phase forPseudomonasphage ɸKZ

et al. 2020

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As part of the ongoing renaissance of phage biology, more phage genomes are becoming available through DNA sequencing. However, our understanding of the transcriptome architecture that allows these genomes to be expressed during host infection is generally poor. Transcription start sites (TSSs) and operons have been mapped for very few phages, and an annotated global RNA map of a phagealone or together with its infected host-is not available at all. Here, we applied differential RNA-seq (dRNA-seq) to study the early, host takeover phase of infection by assessing the transcriptome structure of Pseudomonas aeruginosa jumbo phage ɸKZ, a model phage for viral genetics and structural research. This map substantially expands the number of early expressed viral genes, defining TSSs that are active ten minutes after ɸKZ infection. Simultaneously, we record gene expression changes in the host transcriptome during this critical metabolism conversion. In addition to previously reported upregulation of genes associated with amino acid metabolism, we observe strong activation of genes with functions in biofilm formation (cdrAB) and iron storage (bfrB), as well as an activation of the antitoxin ParD. Conversely, ɸKZ infection rapidly down-regulates complexes IV and V of oxidative phosphorylation (atpCDGHF and cyoABCDE). Taken together, our data provide new insights into the transcriptional organization and infection process of the giant bacteriophage ɸKZ and adds a framework for the genome-wide transcriptomic analysis of phage-host interactions.

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“…Major RNA-binding proteins and associated ncRNAs P. aeruginosa is known to possess two large post-transcriptional networks that depend on globally acting RBPs: one governed by Hfq, the other by two CsrA-like Rsm proteins. The mRNA and ncRNA ligands of these RBPs have been mapped in a transcriptome-wide fashion (8,9,17,19), albeit not in the growth phase studied here.…”

Section: Topology Of the Pseudomonas Transcriptomementioning

confidence: 96%

A Grad-seq view of RNA and protein complexes inPseudomonas aeruginosaunder standard and bacteriophage predation conditions

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Wicke

Chihara

et al. 2020

Preprint

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The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and post-transcriptional regulation through the RNA-binding proteins Hfq, RsmA and RsmN have been studied in detail, global information about stable RNA–protein complexes is currently lacking in this human pathogen. Here, we implement Gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated pRNAs. We observe specific clusters of non-coding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased co-sedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded non-coding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.IMPORTANCEStable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbour some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong co-localization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

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Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

Cited by 22 publications

References 70 publications

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

Introducing differential RNA-seq mapping to track the early infection phase forPseudomonasphage ɸKZ

A Grad-seq view of RNA and protein complexes inPseudomonas aeruginosaunder standard and bacteriophage predation conditions

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