XendoU is required for calcium-mediated RNA degradation in vitro. (A) Recombinant wild-type XendoU was incubated with increasing concentrations of CaCl₂, MgCl₂, MnCl₂, and total RNA from egg extract. RNA was visualized on a denaturing acrylamide gel stained with SYBR Green II. (B) Recombinant H162A and K224A were incubated with metal and RNA and visualized as in A. A very faint band (arrow) appears in the presence of calcium and K224A. (C) XendoU or mock-depleted (IgG) cytosol was incubated with 1 mM CaCl₂. Recombinant wild-type XendoU (rXU) was added to XendoU-depleted, mock-depleted (IgG), or undepleted cytosol and analyzed as in A. (D) Western blot of XendoU demonstrating the antibody-mediated depletion in CSF extract as compared with undepleted (CSF) or mock-depleted (IgG) extracts. (E) Cytosol or cytosol mixed with light membranes were incubated with increasing concentrations of CaCl₂, and RNA was visualized as in A.