Functional and Biochemical Characterization of Dib1's Role in Pre-Messenger RNA Splicing
The spliceosome is a dynamic macromolecular machine that undergoes a series of conformational rearrangements as it transitions between the several states required for accurate splicing. The transition from the B to Bact is a key part of spliceosome assembly and is defined by the departure of several proteins, including essential U5 component Dib1. Recent structural studies suggest that Dib1 has a role in preventing premature spliceosome activation, as it is positioned adjacent to the U6 snRNA ACAGAGA and the U5 loop I, but its mechanism is unknown. Our data indicate that Dib1 is a robust protein that tolerates incorporation of many mutations, even at positions thought to be key for its folding stability. However, we have identified two temperature-sensitive mutants that stall in vitro splicing prior to the first catalytic step and block assembly at the B complex. In addition, Dib1 readily exchanges in splicing extracts despite being a central component of the U5 snRNP, suggesting that the binding site of Dib1 is flexible. Structural analyses show that the overall conformation of Dib1 and the mutants are not affected by temperature, so the temperature sensitive defects most likely result from altered interactions between Dib1 and other spliceosomal components. Together, these data lead to a new understanding of Dib1's role in the B to Bact transition and provide a model for how dynamic protein–RNA interactions contribute to the correct assembly of a complex molecular machine.
Document Object Identifier (DOI)
Schreib, C. C., Bowman, E. K., Hernandez, C. A., Lucas, A. L., Potts, C. H. S., & Maeder, C. (2018). Functional and biochemical characterization of Dib1's role in pre-messenger RNA splicing. Journal of Molecular Biology, 430(11), 1640-1651. doi:10.1016/j.jmb.2018.04.027