• UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts.

UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts.

Plant methods (2016-11-09)
Zhicheng Zhang, Kurt Boonen, Piero Ferrari, Liliane Schoofs, Ewald Janssens, Vera van Noort, Filip Rolland, Koen Geuten

The 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 We 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) The 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.

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Yttrium aluminum oxide, nanopowder, <150 nm particle size (TEM), 99% trace metals basis