Dataset Information

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli.

ABSTRACT: Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ?85% of all E. coli transcripts and ?49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

SUBMITTER: Hor J

PROVIDER: S-EPMC7498339 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli.

Hör Jens J Di Giorgio Silvia S Gerovac Milan M Venturini Elisa E Förstner Konrad U KU Vogel Jörg J

Nucleic acids research 20200901 16

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the prote ...[more]

PMID: 32813020

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Project description: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 posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. 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 product RNAs (pRNAs). We observe specific clusters of noncoding 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 cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding 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.IMPORTANCE Stable 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 harbor 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 colocalization 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.

Dataset Information

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli.

Publications

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli.

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