Eukaryotic precursor-messenger RNAs (pre-mRNAs) undergo extensive compositional pre-processing to become mature, protein-coding mRNAs. Among the vital transformations underlying pre-mRNA maturation, the 3′-end processing machinery (3EPM) orchestrates 3′-end pre-mRNA cleavage via a yet elusive catalytic mechanism. Here, all-atom simulations of a 350,000 atom model of 3EPM disclose that its catalytic engine, the CPSF73 endonuclease, cleaves the 3′-end of pre-mRNA via an associative two-Zn2+-ion-aided mechanism, where the metals, besides activating the nucleophile and stabilizing the transition state, as in canonical two-Mg2+-ion catalysis, assist the leaving group's protonation. In spite of the distinctive metal type content, an in depth structural and mechanistic inspection of two-Zn2+-ion- versus two-Mg2+-ion-dependent nucleases unlocks striking similarities between the expanded positive charge of their catalytic motifs, with the metals being assisted by second-shell basic residues/metal ions. This catalytic architecture, hence, emerges as a critical prerequisite for a common and effective mechanism of phosphodiester bond hydrolysis in nuclease enzymes. Ostensibly, our outcomes unveil the salient molecular traits of the 3EPM mechanism, providing tantalizing opportunities in harnessing this emerging drug target to fight the wide variety of human diseases associated with a deregulated pre-mRNA processing.
