UV-crosslinking and interactome capture

In vivo crosslinking and isolation of Arabidopsis RBPs was performed, as detailed described³¹. Briefly, each flask of cells was divided into two, to approximately one gram of cells per biological replicate. Cells were placed onto Petri dishes and one of each pair was used for UV-crosslinking (UV) and the other for the control (no UV-crosslinking (nUV)). Cells for UV-crosslinking were irradiated with 0.15 J/cm² UV light at 254 nm for ~90 s, lysed in lysis buffer (20 mM Tris (pH 7.5), 500 mM LiCl, 0.5% (w/v) lithium dodecyl sulfate, 1 mM EDTA, 5 mM DTT) using a PowerGen 125 grinder (ThermoFisher Scientific, Rockford, IL, USA), vortexed for 1 min and incubated on ice for 10 min. Debris were pelleted by using an Allegra^® X-22R centrifuge (Beckman Coulter Corp., Brea, CA, USA) at 400 × g for 10 min at 4 °C and the supernatant was carefully collected. Oligo(dT)₂₅ magnetic beads (NEB, S1419S) were used to capture poly(A)⁺ RNAs, undergo several washes prior to elution of mRNA-protein complexes, as described elsewhere³¹. To assess the quality of the mRNA-protein crosslinked complex pull down, an additional control was performed by treating the sample with RNase T1/A mix (ThermoFisher Scientific) and the reaction was performed according to the manufacturer’s recommendation. For RBP analysis samples were treated with RNase A/T1 mix to release them from the captured RNA molecules. Proteins were then analyzed 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 the manufacturers’ recommendations.