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Chemical crosslinking extends and complements UV crosslinking in analysis of RNA and DNA nucleic acid protein interactions by mass spectrometry

ABSTRACT: UV (ultra violet) crosslinking with mass spectrometry (XL MS) has been established for identifying RNA and DNA binding proteins along with their domains and amino acids involved. Here, we explore chemical XL MS, for RNA protein, DNA protein and nucleotide protein complexes in vitro and in vivo. We also introduce a novel nucleotide protein crosslink search engine, NuXL, for fast and robust identification of such crosslinks at amino-acid resolution. Chemical XL MS complements and extends UV XL MS by generating different crosslink species. The added spatial information facilitates integrative structural modelling of nucleic acid protein complexes. In vivo UV and chemical XL MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid protein crosslink inventory at amino acid level. RNA crosslinks cover most RNA binding proteins DNA and RNA crosslinks are found in transcriptional repressors and activators. Our workflow adds spatial information to the RNA binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor binding domains.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli (ncbitaxon:562) Homo Sapiens (ncbitaxon:9606) Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: Prof. Henning Urlaub

PROVIDER: MSV000094879 | MassIVE | Tue May 28 00:31:00 BST 2024

SECONDARY ACCESSION(S): PXD052614

REPOSITORIES: MassIVE

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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.

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Chemical crosslinking extends and complements UV crosslinking in analysis of RNA and DNA nucleic acid protein interactions by mass spectrometry

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