Prediction of motifs involved in NCL-miRNA interactions
NCL-miRNA interactions were investigated using the protein-RNA docking software HDOCK [92] and NPDock [93]. Both programs employ a rigid body docking approach followed by scoring and clustering of structures with the lowest minimum energy followed by refinement of the results through Monte Carlo simulations. Both the programs are considered the top ranked algorithms for protein-RNA docking and score well in benchmarks. The docking results were visualized in PyMOL [94], and the interactions mapped out using Nucplot from PDBSum [95]. The key interacting residues in the NCL-RBDs were identified based on the 10 top scoring scenarios from each docking run. These residues were compared with putative residues of NCL-RBDs identified using catRAPID [96], a program that predicts binding propensity of a protein sequence to a given RNA sequence by measuring physicochemical properties and shape complementarity. Additionally, control docking experiments were conducted with miRNA models (mir-155) which has been shown not to interact with NCL [41]. Similarly, docking experiments were performed using in silico mutagenesis at key residues of NCL identified to be important in NCL-miRNA interactions in the current study (Y219A, R261A, F311A).