Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

1-POS Board 1 MONOVALENT METAL IONS IN PRE-MRNA SPLICING Jana Aupic 1 ; Jure Borišek 2 ; Sebastian M Fica 3 ; Wojciech P Galej 4 ; Alessandra Magistrato 1 ; 1 CNR-IOM c/o International School for Advanced Studies (SISSA), Trieste, Italy 2 National Institute of Chemistry, Theory department, Ljubljana, Slovenia 3 University of Oxford, Department of Biochemistry, Oxford, United Kingdom 4 European Molecular Biology Laboratory, Grenoble, France Splicing, i.e. excision of non-coding introns and ligation of coding exon regions, is an integral step of messenger RNA (mRNA) maturation. The splicing reaction is catalyzed by the spliceosome, an intricate and highly dynamic ribonucleoprotein complex that relies on a two Mg 2+ -ion motif to perform phosphodiester bond breakage and formation. Cryogenic electron microscopy (cryo-EM) studies of the spliceosome complex furnished unprecedented insights into the functional mechanism of splicing, identifying conformational and compositional changes that govern the process. Conversely, the complete classification of all metal cofactors is still an ongoing challenge, despite their relevance for spliceosome folding and catalysis. All-atom molecular dynamics (MD) simulations, particularly in the hybrid quantum-classical (QM/MM) regime, can importantly complement cryo-EM data by distinguishing the precise location and identity of metal ions and disentangling their dynamic and functional behavior along the splicing cycle. Here, we employ QM/MM MD to characterize the role of second shell K + ions in splicing. We show that the K + ion positioned in the direct vicinity of the two-Mg 2+ -ion motif promotes the first splicing reaction by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Furthermore, an additional dynamic K + ion, present during the second splicing step, is paramount for 3’ splice site selection, governing the correct positioning of the intron-exon boundary at the active site. Taken together, our findings suggest monovalent ions play an instrumental and multifaceted role in RNA processing.

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