The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3’-splice-site (3’SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3’SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing.

Intron splicing of a nascent mRNA transcript by spliceosome (SPL) is a hallmark of gene regulation in eukaryotes. SPL is a majestic molecular machine composed of an entangled network of proteins and RNAs that meticulously promotes intron splicing through the formation of eight intermediate complexes. Cross-communication among the critical distal proteins of the SPL assembly is pivotal for fast and accurate directing of the compositional and conformational readjustments necessary to achieve high splicing fidelity. Here, molecular dynamics (MD) simulations of an 800 000 atom model of SPL C complex from yeast Saccharomyces cerevisiae and community network analysis enabled us to decrypt the complexity of this huge molecular

The spliceosome, a protein-directed metallo-ribozyme, catalyzes premature mRNA splicing via two transesterification reactions. The atomic-level details of the splicing mechanism and the role of the entwined protein-RNA environment during catalysis remain unresolved. Here, quantum-classical molecular dynamics simulations along with thermodynamic integration unveil that the second catalytic (exon-ligation) step occurs via an associative two-Mg2+-ion mechanism, exclusively catalyzed by RNA, with the scissile phosphate mediating a proton transfer from the nucleophile to the leaving group. Our outcomes provide fundamental advances in understanding the splicing mechanism in eukaryotes disclosing how the catalytic core of spliceosome’s ancestors self-splicing ribozymes remained conserved during evolution.