PXD049303

PXD049303 is an original dataset announced via ProteomeXchange.

Title	Revealing the Arabidopsis AtGRP7 mRNA interactome by specific RNP capture
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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:43:38.465.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marlene Reichel
SpeciesList	scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2024-02-09 07:27:49	ID requested
1	2024-06-04 04:14:15	announced
⏵ 2	2024-10-22 06:43:38	announced	2024-10-22: Updated project metadata.

10.1101/2024.04.04.588066;

submitter keyword: AtGRP7,Arabidopsis, RNA-binding protein, mRNA interactome

Dorothee Staiger
contact affiliation	Bielefeld University, Faculty of Biology, RNA biology and molecular physiology
contact email	dorothee.staiger@uni-bielefeld.de
lab head
Marlene Reichel
contact affiliation	Copenhagen University
contact email	marlene.reichel@bio.ku.dk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD049303
     1. Label: PRIDE project
     2. Name: Revealing the Arabidopsis AtGRP7 mRNA interactome by specific RNP capture