3D structural models of NCL RBD1-4, RBD3-4 and specific miRNAs provide a complete structural picture needed to study NCL-miRNA interactions
The tandem pair of NCL RBD1-2 structure is experimentally resolved (PDB ID: 2KRR) [39] and well characterized for its interactions with various rRNA [16] and mRNAs [25,26]. While individual crystal structures of RBD3 and RBD4 are available, albeit misannotated (PDB IDs: 2FC9 & 2FC8 [40]), the functional role(s) of the NCL RBD3-4 tandem pair remain unresolved and unexplored. Structural information for all the RBDs in NCL are critical to fully understand the mechanistic details about various RNA targets of NCL and the key features that render target-specificity. Lack of structural details on RBD1-4 as a whole and the RBD3-4 tandem pair, pose limitations in dissecting the specificity with which NCL binds to a wide-range of RNA species and plays pivotal role/s in pathophysiology. Therefore, structural models of NCL RBDs and miRNA molecules generated using template based and ab initioapproaches were analyzed to identify robust models with high evaluation profiles (Supporting Tables S1 and S2 ) to bridge the gap in NCL RBD structural information. Fig 1 shows that the superposition of the top ranked RBD1-4 (Fig 1A ) and RBD3-4 (Fig 1B ) models with the existing individual crystal structures of RBD1, 2, 3 and 4 overlays very well with minor deviations in the loop regions.
Previous biochemistry studies have shown that NCL interacts with pri-mir-15a, pri-mir-16-1, pri-mir103a, pri-mir-21, pri-mir-221, and pri-mir-222 [21,41]. Structural information of these miRNA molecules is largely unavailable in the current databases. Therefore, 3D miRNA models generated based on secondary structure prediction using 3D modeling programs were evaluated to identify top ranked models (Fig 2 ). Additionally, if multiple models were ranked the same in structural evaluation profiles (Supporting Table S3 ), they were all used in RNA-Protein docking analysis to check for reproducibility of results. Our predicted models provide a complete picture of the structural information for both NCL and the miRNA analyzed in this study.