Atomic-Level Mechanism of Pre-mRNA Splicing in Health and Disease.

Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.