Atomistic and Coarse-Grained MD Simulation Studies of the Energetics and Interactions of Regulatory Dib1 Protein Inside a Highly Dynamic Pre-Catalytic Spliceosomal Macromolecular Complex Preceding Pre-Messenger RNA Splicing in Eukaryotic Cells*

The spliceosome is a complex RNA/protein macromolecular machine responsible for catalyzing the removal of introns from pre-messenger RNA in eukaryotic cells. As part of the highly dynamic spliceosome assembly pathway, the key protein Dib1 must depart the pre-catalytic B complex prior to pre-mRNA splicing. Experimentally, we have identified several Dib1 temperature-sensitive mutants that disrupt splicing activity and spliceosome assembly. However, the molecular mechanisms underlying how these mutations perturb protein-protein and protein-RNA interactions in the B complex are not known. Using 200ns of atomistic and 2µs of coarse-grained MD simulations, the interactions between Dib1 and its neighboring molecules were determined for spliceosomal B complexes containing wild-type and mutant Dib1 at conditions similar to biochemical experiments. We observed specific changes in the interaction energy, residue-contact, and hydrogen-bonding propensity at the interfacial region between Dib1 and neighboring RNAs and proteins. Our results offer an explanation for the experimental finding that the Dib1 mutations disrupt the transition from B to B^act complexes in the splicing pathway.