Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

Background The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. 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 oligo(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. Seedlings 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 (UTR). The efficiency of RNA capture was greatly enhanced by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5’UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. 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 (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. Conclusion We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up-scaling the protocol should be applicable for less abundant transcripts. ### Competing Interest Statement The authors have declared no competing interest. * ALBA : ACETYLATION LOWERS BINDING AFFINITY AO : antisense oligonucleotide ASCO : ALTERNATIVE SPLICING COMPETITOR AtGRP7 : Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 BAP : beads after pulldown BBP : beads before pulldown CHART : Capture Hybridization Analysis of RNA Targets EDC-HCl : N -(3-dimethylaminopropyl)- N′ -ethylcarbodiimide hydrochloride eRIC : enhanced RNA interactome capture gDNA : genomic DNA GFP : Green fluorescent protein GO : Gene ontology h : hour iCLIP : individual-nucleotide resolution crosslinking and immunoprecipitation IN : input LD : light-dark LL : continuous light LNA : locked nucleic acid lncRNA : long noncoding RNA MALAT1 : metastasis-associated lung adenocarcinoma transcript 1 MES : 2-(N-morpholino)ethanesulfonic acid min : minute MS : Mass spectrometry NEAT1 : nuclear-enriched abundant transcript 1 NSRa : NUCLEAR SPECKLE RNA-BINDING PROTEIN a PBS : phosphate buffered saline PCR : Polymerase chain reaction PHAROH : Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC PVP40 : polyvinylpyrrolidone 40 PWB : protein wash buffer RBPome : RNA binding proteome RIC : RNA interactome capture RNP : Ribonucleoprotein RT : reverse transcription SN : supernatant snRNA : small nuclear RNA UTR : untranslated region UV light : ultraviolet light