Only few computational studies of ion-dependant RNases mechanisms are found in the literature, almost exclusively on RNase H, and several points of its mechanism remain debated, including for instance the initial state-deprotonated or not-of the nucleophilic water molecule, the proton shuttle pathway(s), and the identity of a possible general acid to reprotonate the 3 ′ OH leaving group. The recently obtained structures of the rRNA:M5 complex thus appeared as an opportunity to examine the molecular mechanism of a different 2-ion dependent RNase.