UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts.
BACKGROUND
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 forArabidopsis thalianaleaf mesophyll protoplasts, a cell type often used in functional cellular assays.


RESULTS
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)25bead 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.


CONCLUSIONS
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.
Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Louvain, Belgium.
Zhicheng
Z
Zhang
Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Louvain, Belgium.
Kurt
K
Boonen
Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200d, 3001 Louvain, Belgium.
Piero
P
Ferrari
Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Louvain, Belgium.
Liliane
L
Schoofs
Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200d, 3001 Louvain, Belgium.
Ewald
E
Janssens
Department of Microbial and Molecular Systems, KU Leuven, Kasteelpark Arenberg 22, 3001 Louvain, Belgium.
Vera
V
van Noort
Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Louvain, Belgium.
Filip
F
Rolland
Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Louvain, Belgium.
Koen
K
Geuten
Arabidopsis thaliana leaf mesophyll protoplasts; In vivo UV crosslinking; Messenger RNA-binding proteins; Messenger ribonucleoprotein complexes; mRNA-bound proteome