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.