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Specific RNP capture with antisense LNA/DNA mixmers.

ABSTRACT: RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

SUBMITTER: Rogell B

PROVIDER: S-EPMC5513073 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Specific RNP capture with antisense LNA/DNA mixmers.

Rogell Birgit B Fischer Bernd B Rettel Mandy M Krijgsveld Jeroen J Castello Alfredo A Hentze Matthias W MW

RNA (New York, N.Y.) 20170505 8

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular ...[more]

PMID: 28476952

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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.

Dataset Information

Specific RNP capture with antisense LNA/DNA mixmers.

Publications

Specific RNP capture with antisense LNA/DNA mixmers.

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