In Vivo RBP Crosslinking and Isolation in Arabidopsis

In vivo UV-crosslinking and isolation of Arabidopsis RBPs was performed, as previously described [12 (link)], using a protocol that utilizes a modified method originally optimized for HeLa cells [11 (link)]. Sample from each time-point were split into two, one set for UV-crosslinking and the second set for non UV-crosslinking. Samples for UV-crosslinking were irradiated in vivo with UV (254 nm) and the mRNA-protein complexes were pulled down using oligo(dT) beads. Purified proteins were analyzed by label free tandem mass spectrometry. Similarly to [12 (link)], the quality of the mRNA-protein crosslinked complex pull-down was assessed by performing an additional control whereby the sample was treated with RNase T1/A mix (Thermo-Fisher Scientific) according to the manufacturer’s recommendations. To isolate RBPs, mRNA-protein samples were treated with RNase A/T1 mix to release them from the captured RNA molecules. Crosslinking and isolation of RBPs were evaluated by western blotting using antibodies against polypyrimidine tract-binding protein 1, β-actin (Sigma Aldrich, St Louis, MO, USA) and histone 3 (Abcam, Cambridge, UK) following manufacturer’s recommendations (see [12 (link)]).