Dataset Information

The RNA fold interactome of evolutionary conserved yeast RNA structures in S. cerevisiae

ABSTRACT: Based on previous DMS-Seq data that provides in vivo structural conformation of RNA, we here applied a streamlined RNA pull-down approach to study 186 evolutionary conserved RNA folds in S. cerevisiae. These folds bound 162 different proteins, with the majority known or inferred to be RNA-binding proteins. In contrast to global RNA interactome capture methods; we were able to associate individual messenger RNAs harboring conserved RNA folds with their set of bound regulating proteins. Our data suggests a more widespread function of tRNA synthetases as mRNA regulators. Using an in vivo RNA reporter screen, we show that specific mRNA-RBP pairs regulate protein expression, which connects structural RNA features recognized by the RNA-binding protein to functionality.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: F Butter

LAB HEAD: Falk Butter

PROVIDER: PXD014092 | Pride | 2020-07-02

REPOSITORIES: Pride

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			Action	DRS
	20160410_QEP2_FBU_NC_RNAlib_P2A1_for_160410205005.raw	Raw
	20160410_QEP2_FBU_NC_RNAlib_P2A1_rev.raw	Raw
	20160410_QEP2_FBU_NC_RNAlib_P2A2_for.raw	Raw
	20160410_QEP2_FBU_NC_RNAlib_P2A2_rev.raw	Raw
	20160410_QEP2_FBU_NC_RNAlib_P2A3_for.raw	Raw

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The RNA fold interactome of evolutionary conserved RNA structures in S. cerevisiae.

Casas-Vila Nuria N Sayols Sergi S Pérez-Martínez Lara L Scheibe Marion M Butter Falk F

Nature communications 20200603 1

RNA-binding proteins play key roles in regulation of gene expression via recognition of structural features in RNA molecules. Here we apply a quantitative RNA pull-down approach to 186 evolutionary conserved RNA structures and report 162 interacting proteins. Unlike global RNA interactome capture, we associate individual RNA structures within messenger RNA with their interacting proteins. Of our binders 69% are known RNA-binding proteins, whereas some are previously unrelated to RNA binding and ...[more]

PMID: 32493961

Publication: 1/2

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Project description:Background The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA pro-cessing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oli-go(dT) beads and the RNA binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. Results Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seed-lings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5´untranslated region. The efficiency of RNA capture was greatly enhanced by the use of locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. In total, we identified more than 300 proteins interacting with the AtGRP7 RNA. These were benchmarked against proteins pulled down by AtGRP7 in vitro transcripts from nuclear lysates. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation. The circadian expression of the AtGRP7 transcript was slightly changed in an alba loss-of-function mutant compared to wild-type, demonstrating the functional relevance of the interaction. Conclusion We adapted specific RNP capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up-scaling of the protocol should be applicable for less abundant transcripts. Continuous improvements of mass spectrometry sensitivity will also help to increase the range of proteins that can be identified.