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

Hör, Jens; Giorgio, Silvia Di; Gerovac, Milan; Venturini, Elisa; Förstner, Konrad U.; Vogel, Jörg

doi:10.1101/2020.06.29.177014

Cited by 3 publications

(2 citation statements)

References 104 publications

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“…Interestingly, ecHrpA was shown to interact with ribosomal proteins (59). Furthermore, Salmonella enterica HrpA has been found to associate with the small ribosomal subunit (60), and a recent GradSeq analysis in E. coli revealed an association of ecHrpA with 70S ribosomes or polysomes (61). It is conceivable that an interaction of HrpA with ribosomes, mediated, for example, via the CTR, directly alters the effect of translation inhibitors, while the effect of HrpA on other antibiotics may result from HrpAribosome/polysome interactions that elicit changes in the levels of certain proteins.…”

Section: Discussionmentioning

confidence: 99%

Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility

Grass

Wollenhaupt

Barthel

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5′ RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.

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Section: Discussionmentioning

confidence: 99%

Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility

Grass

Wollenhaupt

Barthel

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…These studies demonstrate that small protein genes are much more abundant than previously imagined. For instance, more than 100 sORFs have been experimentally verified in the model organism E. coli ( 3 , 4 , 12 , 14 , 15 ). An extensive metagenomics study of the human microbiome identified more than 4,000 putative small protein families, indicating a hidden world of small proteins awaiting to be explored ( 10 ).…”

Section: Introductionmentioning

confidence: 99%

The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR

Andresen

Martínez-Burgo

Zangelin

et al. 2020

mBio

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Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.

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

Chihara

Gerovac²,

Hör³

et al. 2022

Preprint

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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.

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

Cited by 3 publications

References 104 publications

Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility

Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility

The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR

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

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