Abstract

BackgroundRNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level.ResultsLabel-free mass spectrometry enabled the identification 567 proteins of which 150 significantly responded to the drought-induced treatment. A gene ontology analysis revealed enrichment in the “RNA binding” and “RNA processing” categories as well as biological processes such as “response to abscisic acid” and “response to water deprivation”. Importantly, a large number of the stress responsive proteins have not previously been identified as RBPs and include proteins in carbohydrate metabolism and in the glycolytic and citric acid pathways in particular. This suggests that RBPs have hitherto unknown roles in processes that govern metabolic changes during stress responses. Furthermore, a comparative analysis of RBP domain architectures shows both, plant specific and common domain architectures between plants and animals. The latter could be an indication that RBPs are part of an ancient stress response.ConclusionThis study establishes mRNA interactome capture technique as an approach to study stress signal responses implicated in environmental changes. Our findings denote RBP changes in the proteome as critical components in plant adaptation to changing environments and in particular drought stress protein-dependent changes in RNA metabolism.

Highlights

  • RNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression

  • Interactome capture involves in vivo fixing of proteins to their target mRNAs by Ultra violet (UV) crosslinking followed by purification of mRNA-protein complexes through affinity capture of polyadenylated RNA and analyzing interacting proteins by tandem mass spectrometry (MS/MS)

  • Abscisic acid assay To confirm whether the treatment of cell suspension cultures with 40% (v/v) polyethylene glycol (PEG) was sufficient to induce or mimic drought stress, we performed an ABA assay using the Phytodetek® ABA Immunoassay kit

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Summary

Introduction

RNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression. Interactome capture involves in vivo fixing of proteins to their target mRNAs by UV crosslinking followed by purification of mRNA-protein complexes through affinity capture of polyadenylated RNA and analyzing interacting proteins by tandem mass spectrometry (MS/MS) This technique has a great advantage over other crosslinking techniques based on chemical fixation in that it generates covalent linkages between physically interacting proteins and mRNAs in vivo [20, 21]. It permits time resolved isolation of RBPs allowing characterization of targeted developmental and physiological states of cellular systems. We suggest that stress-induced RBPs may be an evolutionarily conserved mechanism governing post-transcriptional responses to stress

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