2.11 Biotin RNA Pull-Down Assay
RNA pull-down assay was performed as previously described (Hu, Jin, Xu, Wang, Thorne, Zhang & Wu, 2018). Biotin-labeled CRIR1 orAnti-CRIR1 RNAs were transcribed in vitro using the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Invitrogen), and purified with RNeasy Mini Kit (Qiagen). Total protein was extracted from 3-week-old cassava seedlings using Total Protein Extraction Kit for plant tissues (Invent Biotechnologies, Inc) and pre-cleared against streptavidin magnetic beads (Invitrogen). Biotin-labeled CRIR1 RNAs adsorbed to streptavidin magnetic beads were then incubated with pre-cleared protein extract in the presence of complete protease inhibitors cocktail, phosphatase inhibitor cocktail, and RNase inhibitors, at 4 °C for 12 h, followed by washing five times in wash buffer (100 mM KCl, 50 mM Tris (pH 7.4), 0.5 mM dithiothreitol, 0.5% NP-40, 1mM DTT and RNase inhibitors) and elution in 5×SDS sample loading buffer. Eluted proteins were separated by SDS-PAGE and were then subjected to mass spectrometric analysis.