R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation

Caudron-Herger, Maïwen; Rusin, Scott F.; Adamo, Mark E.; Seiler, Jeanette; Schmid, Vera K.; Barreau, Elsa; Kettenbach, Arminja N.; Diederichs, Sven

doi:10.1016/j.molcel.2019.04.018

Cited by 98 publications

(106 citation statements)

References 63 publications

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“…Recently, very interesting orthogonal approaches have been developed to study RNA-protein interactions, for example R-DeeP, a density gradient centrifugation method that compares sedimentation of protein complexes before and after RNase treatment (Caudron-Herger et al, 2019). R-DeeP allows to quantify the fraction of an RBP that co-sediments with RNA and should therefore be well suited to assess RNA-binding activities for individual RBPs.…”

Section: Discussionmentioning

confidence: 99%

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Kilchert

Kecman

Priest

et al. 2019

Preprint

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Abbreviations: gene ontology (GO); messenger RNA (mRNA); mass spectrometry (MS); polyadenylation site (PAS); RNA-binding domain (RBD); RNA-binding protein (RBP);RNA interactome capture (RIC); ribonucleoprotein particle (RNP); ribosomal protein (RP); transcription elongation factor (TEF); wild-type (WT); whole cell extract (WCE); cleavage and polyadenylation factor (CPF); cleavage factors IA and IB (CFIA and CFIB) 2 Abstract Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNAbinding activity and provide key information about the RNA-binding properties of large multiprotein complexes, such as the mRNA 3' end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

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Section: Discussionmentioning

confidence: 99%

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Kilchert

Kecman

Priest

et al. 2019

Preprint

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“…RBPs will then be identified by comparative mass spectrometry of the treated and untreated gradients, so the theory. As do the conceptually related R-DeeP (Caudron-Herger et al 2019) and DIF-FRAC (Mallam et al 2019) approaches for RBP discovery in eukaryotes, GradR works without a UVcrosslinking step.…”

Section: Differential Sedimentation Profiles Of Rbps Upon Prior Rnasementioning

confidence: 99%

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

Gerovac

Mouali

Kuper

et al. 2020

RNA

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RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinOdomain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.

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“…For the preparation of lysates for the testing of RNA dependency through ultracentrifugation, previously published protocols were used as a basis (38,39). HeLa cells were cultured on a 15 cm dish and lysed in 300 µl lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM KCl, 0.5% NP-40, 2 mM EDTA, 1 mM NaF, 0.5 mM DTT, protease inhibitor).…”

Section: Sucrose Density Centrifugation and Fractionationmentioning

confidence: 99%

RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1

Huppertz

Perez-Perri

Mantas

et al. 2020

Preprint

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Cells must coordinate their metabolism and fate trajectories (1, 2), but the underlying mechanisms are only beginning to be discovered. To understand why the glycolytic enzyme enolase 1 (ENO1) binds RNA (3-6), we studied this phenomenon in vitro, in human cells, and during mouse embryonic stem cell differentiation. We find specific cellular RNA ligands that inhibit ENO1's enzymatic activity in vitro. Increasing the concentration of these ligands in cultured cells inhibits glycolysis. We demonstrate that pluripotent stem cells expressing an ENO1 mutant that is hyper-inhibited by RNA are severely impaired in their glycolytic capacity and in endodermal differentiation, whereas cells with an RNA binding-deficient ENO1 mutant display disproportionately high endodermal marker expression. Our findings uncover ENO1 riboregulation as a novel form of metabolic control. They also describe an unprecedented mechanism involved in the regulation of stem cell differentiation.

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R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation

Cited by 98 publications

References 63 publications

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1

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