Figure 6. Recombinant core RNA exosome and individual subunits stimulate AID activity.

(A) Coomassie blue stained polyacrylamide gel electrophoreisis analysis of the 9 subunit recombinant RNA exosome complex generated from individual subunits. (B) Comparative analysis of the ability of RNA exosome complex from 293T cells (Exo²⁹³) and recombinant RNA core exosome complex (rExo) to stimulate AID deamination activity as measured by ³H release/SHM substrate assay in Fig. 5A. See Supp. Methods and Fig. S6 for details. Optimal amounts of RNA exosome^293T and recombinant RNA exosome core complex were used (Fig. S6; Supp. Methods). (C) Assay of recombinant exosome complex to stimulate strand-specific AID deamination of a transcribed SHM substrate via assay in Fig. 5C. Non-template and template strand deamination are shown on left and right panels, respectively. Reactions contained either AID, recombinant core RNA exomsome (rExo^9wt) or both (AID + rExo^9wt) as indicated. (D) Individual recombinant RNA exosome subunits were assayed for ability to promote AID deamination of a T7-transcribed SHM substrate as outlined in Fig. 5A. Added exosome components are indicated. Control reactions with AID alone or AID plus T7 polymerase are on the left. A positive control with complete recombinant core RNA exosome (rExo⁹) plus T7 and AID is on the right. For panels B and D, values represent the average and standard deviation from the mean for three independent experiments. See also Figure S 6&7.