Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome

Smith, Tom; Villanueva, Eneko; Queiroz, Rayner M L; Dawson, Charlotte S.; Elzek, Mohamed; Urdaneta, Erika C.; Willis, Anne E.; Beckmann, Benedikt M.; Krijgsveld, Jeroen; Lilley, Kathryn S.

doi:10.1016/j.cbpa.2020.01.009

Cited by 38 publications

(36 citation statements)

References 56 publications

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“…This study brought to three the number of ProQ/FinO-like chaperones simultaneously residing in the same bacterial cell (ProQ, FinO, FopA), which is an exceptional case with potentially interesting evolutionary implications. All in all, these approaches beautifully complement the existing RBP discovery techniques, such as RNA interactome capture and the new crosslinking-based methods OOPS, PTex, TRAPP, and XRNAX (Hentze et al, 2018;Queiroz et al, 2019;Shchepachev et al, 2019;Trendel et al, 2019;Urdaneta et al, 2019;Smith et al, 2020b), by adding those RNP components which do not interact with RNA directly or crosslink poorly yet contribute to the structure and the functionality of the complexes. Like the classical Grad-seq, they provide important biochemical data on the organization and the RNA dependency of cellular RNPs.…”

Section: Beyond Grad-seqmentioning

confidence: 98%

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

Gerovac

Vogel

Smirnov

2021

Front. Mol. Biosci.

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Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

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Section: Beyond Grad-seqmentioning

confidence: 98%

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

Gerovac

Vogel

Smirnov

2021

Front. Mol. Biosci.

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“…RBPs and study RNA-protein complexes and interactions on a global level (27), one of which is gradient profiling by sequencing, a.k.a. Grad-seq (22).…”

Section: The Past Couple Of Years Have Witnessed the Development Of Nmentioning

confidence: 99%

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

Gerovac

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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“…Systemweite Anreicherung von RNA-Protein-Komplexen XRNAX) [8]. Zwei andere Ansätze (2C, TRAPP) nutzen eine Festphase zur Anreicherung von RNPs durch Bindung von Silica-Beads an Nukleinsäuren [9], während die Techniken R-deep, DIF-FRAC und GradR auf einer Ultrazentrifugation mit Dichtegradienten als Prinzip zur Anreicherung von Komplexen basieren [10] [2,11].…”

Section: Rna-protein-wechselwirkungunclassified

Systemweite Anreicherung von RNA-Protein-Komplexen

Pfister

Beckmann

2020

Biospektrum

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RNA-protein complexes (RNPs) are key players in cell physiology, especially in the context of RNA metabolism. However, their analysis has been dependent on specific protein epitopes or RNA sequence elements, preventing unbiased and cell-wide studies. We developed a three-step protocol, termed Phenol Toluol extraction (PTex), to isolate the complete suite of RNPs in cells, solely based on their unique physicochemical properties after UV-cross-linking. PTex, along with other recently developed unbiased techniques, has the potential to guide the way to cell-wide analysis of RNA-protein interactions.

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Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome

Cited by 38 publications

References 56 publications

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

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

Systemweite Anreicherung von RNA-Protein-Komplexen

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