Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles
Preprint
- 3 October 2021
- preprint
- research article
- Published by Cold Spring Harbor Laboratory
Abstract
Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside EVs and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they comprise both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluid, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that these extravesicular RNAs mediate host-induced gene silencing, rather than RNA inside EVs.One-sentence summary: The apoplast of Arabidopsis leaves contains diverse small and long-noncoding RNAs, including circular RNAs, that are bound to protein complexes and are located outside extracellular vesicles.Keywords
Other Versions
- Published version: Version THE PLANT CELL ONLINE, 34, preprints
This publication has 84 references indexed in Scilit:
- miRBase: annotating high confidence microRNAs using deep sequencing dataNucleic Acids Research, 2013
- Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference PathwaysScience, 2013
- Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7The EMBO Journal, 2013
- An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thalianaNucleic Acids Research, 2012
- Fast gapped-read alignment with Bowtie 2Nature Methods, 2012
- miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNAThe EMBO Journal, 2011
- HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminisTHE PLANT CELL ONLINE, 2010
- Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA FormationCell, 2008
- Substrate Recognition and Catalysis by the Exoribonuclease RNase ROnline Journal of Public Health Informatics, 2006
- Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontianaPlant Physiology, 1980