Abstract
BackgroundThe complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.ResultsWe established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts.ConclusionsThe described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0142-6) contains supplementary material, which is available to authorized users.
Highlights
The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research
Results and discussion mRNA interactome capture from leaf mesophyll cells In this study we focus on the mRNA-bound proteome of plant cells, applying the interactome capture method, which was developed for yeast and human cells to plant mesophyll cells, the major type of ground tissue in plant leaves
The first four steps include Arabidopsis leaf mesophyll protoplast isolation (1), in vivo mRNA-protein crosslinking by UV irradiation (2), protoplast lysis under denaturing conditions (3) and messenger ribonucleoprotein complexes (mRNPs) pull-down and purification by oligo-d(T)25 beads (4)
Summary
The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. When considering different RNA metabolic regulation pathways, post-transcriptional regulation of pre-mature mRNAs is important because of RBP binding specificities from mainly mammalian cells have been experimentally studied by use of common in vitro methods such as RNA electrophoretic mobility shift assay (REMSA), protein affinity purification, systematic evolution of ligands by exponential enrichment (SELEX), fluorescence methods and nuclear magnetic resonance spectroscopy (NMR) [4,5,6,7,8]. The first genome-wide mRNA-bound proteome has been characterized for HEK293 and HeLa
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