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

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

PROVIDER: PRJNA641097 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12067300.fastq.gz	Fastqsanger.gz
	SRR12067301.fastq.gz	Fastqsanger.gz
	SRR12067302.fastq.gz	Fastqsanger.gz
	SRR12067303.fastq.gz	Fastqsanger.gz
	SRR12067304.fastq.gz	Fastqsanger.gz

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

Project description:Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading organism with respect to global bacterial protein-protein networks. However, 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. In presenting use cases for this resource to be exploited, we show that its stable association with 30S ribosomes gives the seemingly noncoding RNA RyeG 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 complements existing interactome resources for the primary model bacterium E. coli with valuable RNA/protein complexome information and should facilitate functional discovery in this and many related species.

2020-06-30 | GSE152974 | GEO

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

Project description: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.

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A Grad-seq resource of RNA and protein complexes of Pseudomonas aeruginosa including transcripts of phage ΦKZ

Project description:Grad-seq of Pseudomonas aerugionosa PAO1 at OD 0.3 with and without infection with ΦKZ bacteriophage

2021-01-06 | GSE157708 | GEO

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

Project description:22 total proteome samples from E. coli wild type resolved on a glycerol gradient resulting in 20 fractions and a pellet fraction. A lysate sample is given as input control. Further, 2 affinity purification samples comparing YggL-3xFLAG pulldown vs. the wild type is given. In this case, each sample was run on a gel and the lanes cut into 15 gel pieces each.

2020-08-10 | PXD019900 | Pride

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

Project description:New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ~500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

2022-10-27 | GSE212408 | GEO

Gradient Sequencing (Grad-Seq) of starved HEK293 cells

Project description:Grad-Seq of serum or amino acid starved HEK293 cells

2022-08-04 | GSE129911 | GEO

A Grad-seq resource of RNA and protein complexes of Pseudomonas aeruginosa including transcripts of phage ΦKZ

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A census of RNA/protein complexes in a model Gram-positive bacterium reveals exonuclease-mediated sRNA activation in competence regulation

Project description:RNA-protein interactions crucially underlie many steps of bacterial gene expression including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast with recent progress in Gram-negative bacteria, knowledge about RNA and protein complexes in Gram-positive species remains scarce. Here, we used Grad-seq to draft a landscape of such complexes in Streptococcus pneumoniae, 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 (a.k.a. YhaM) with sRNAs that control bacterial competence. Contrary to expectation, the nucleolytic activity of Cbf1 stabilized these sRNAs, thereby promoting their function as repressors of competence. These results illustrate how this first RNA/protein complexome resource for a Gram-positive species can be utilized to identify new molecular factors in RNA-based regulation of pathways with relevance to bacterial virulence.

2021-01-27 | GSE138732 | GEO

Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production

Project description:Much of our current knowledge about cellular RNA–protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA–protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA–protein and protein–protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.

2021-05-01 | GSE165672 | GEO

Grad-seq Enterococcus faecalis and faecium

Project description:Grad-seq in Enterococcus faecalis V583 and Enterococcus faecium AUS004.

2022-11-28 | PXD031577 | Pride