New Insights into the Dependence of CPEB3 Ribozyme Cleavage on Mn²⁺ and Mg²

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn²⁺ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg²⁺, as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn²⁺ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn²⁺ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn²⁺ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.