Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

22-POS Board 22 AUGMENTING CRYO-EM DATA ON DYNAMIC SYSTEMS WITH ALL-ATOM SIMULATIONS: EXPLORING THE MECHANISTIC CONTRIBUTION OF PRP2 HELICASE TO SPLICING CYCLE PROGRESSION Pavlína Pokorná 1 ; Alessandra Magistrato 1 ; 1 CNR-IOM/SISSA, Trieste, Italy Premature messenger (pre-mRNA) transcripts undergo splicing before becoming mature funcational RNAs. The splicing process is promoted by spliceosome, a large and dynamic macromolecular RNA-protein assembly. Splicing occurs though multiple consecutive steps which are accompanied by significant compositional and conformational changes of the spliceosome. Responsible for driving this spliceosomal remodeling are specific helicase proteins. Here, we focus on transition from the initially assembled state into catalytic competent spliceosome; a process driven by the PRP2 helicase. We employed all-atom molecular dynamics simulations to augment a Cryo-EM study (Schmitzova et al., 2023) by refining all-atom structure of the splicesomal core in the Baqr state and reconstructing its structural dynamics. We simulated two variants of the system on a micro-second time-scale: spliceosome with PRP2 helicase mechanically stalled in its presumably final state, and a modeled system with PRP2 free to process. The simulations predict dynamic interactions between the helicase and spliceosomal proteins responsible for pre-mRNA selection, which were not resolved in the Cryo-EM structure. Analysis of the movement correlation in the simulated systems suggests that the helicase facilitates large-scale structural rearrangements not only by translocating along the pre-mRNA strand, as revealed by the Cryo-EM structure, but also by actively releasing some of the per mRNA-bound splicing factors. For instance,we observe strong coupling between SF3B1 ring opening dynamics and the movements of the helicase's catalytic domain. Further analyzes and simulations are ongoing to gain more robust sampling and understand coupling between the PRP2 helicase action and the catalytic site. Taken together, our simulations extend the static Cryo-EM structure by predicting all-atom-resolution structural ensembles and reconstructing functional dynamics of this highly plastic system.

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