Fig. 7

Chimeric TRIM25 proteins display correlated RNA-binding and ubiquitination activities. a Domain architecture of wild-type TRIM25 with indicated RBD and chimeric homologous sequences introduced from other TRIM proteins as well as scrambled TRIM25 RBD sequence (aligned with Clustal Omega). b Western blot analysis of T7-TRIM25 chimeric proteins. Lanes 1 and 7 show blot of mock transfected HeLa cells. Lanes 2–6, 8, and 9 represent western blot analysis of overexpressed T7-TRIM25, T7-TRIM25/5, T7-TRIM25/21, T7-TRIM25/27, T7-TRIM25ΔRDB, T7-TRIM25RBD_scr, and T7-TRIM25/65, respectively. c Western blot analyses against T7 and DHX9 of pre-let-7a-1 pull-downs with HeLa cell extracts overexpressing T7-TRIM25 or T7-TRIM25ΔRBD. Lanes 1, 4, and 7 represent 4% (40 μg) of the loading controls (input). Lanes 2, 5, and 8 represent bead-only pull-downs. Lanes 3, 6, and 9 show pre-let-7a-1 pull-down. Note that the T7 antibody highlights an unspecific band visible in lanes 3 and 8. d Western blot analyses against T7 and DHX9 of pre-let-7a-1 pull-downs with HeLa cell extracts overexpressing T7-TRIM25/5, T7-TRIM25/21, or T7-TRIM25/27. Lanes 1, 4, and 7 represent 4% (40 μg) of the loading controls (input). Lanes 2, 5, and 8 represent bead-only pull-downs. Lanes 3, 6, and 9 show pre-let-7a-1 pull-down. e Western blot analyses against T7 and DHX9 of pre-let-7a-1 pull-downs with HeLa cell extracts overexpressing T7-TRIM25RBD_scr or T7-TRIM25/65. Lanes 1, 4, and 7 represent 4% (40 μg) of the loading controls (input). Lanes 2, 5, and 8 represent bead-only pull-downs. Lanes 3, 6, and 9 show pre-let-7a-1 pull-down. Note that the T7 antibody highlights an unspecific band visible in lanes 3 and 6. f Graph displaying a positive person correlation coefficient (0.87) between TRIM25 ubiquitination efficiency (obtained by dividing signal intensities of modified protein bands by main protein bands (b)) and pull-down efficiency (obtained by dividing signal intensities of pull-down band to input (c, d, e))