Fig. 1
From: RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination
TRIM25 is a bona fide RNA-binding protein and the PRY/SPRY domain is responsible for binding to RNA. a EMSA analysis of recombinant His-tagged TRIM25 with pre-let-7a-1. Lane 1 represents the loading control. Lanes 2–11 show EMSA with increasing amounts of TRIM25 (200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 ng). b RNA capture assay result for TRIM25. Red line represents peptide enriched in the RNA-bound fraction. Blue lines show peptides depleted from the RNA-bound fraction. c EMSA analysis of recombinant His-tagged TRIM25ΔRBD with pre-let-7a-1. Lane 1 represents the loading control. Lanes 2–11 show EMSA with increasing amounts of TRIM25ΔRBD (200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 ng). d Domain architecture of wild-type TRIM25 and deletion mutants. Relative position of RNA-enriched peptide is shown in red. e Western blot analyses against T7, TRIM25, 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 T7 antibody highlights an unspecific band visible in lanes 3 and 8. f Western blot analyses against T7 and DHX9 of pre-let-7a-1 pull-downs with HeLa TRIM25 KO cell extracts overexpressing T7-TRIM25, T7-TRIM25ΔRBD, or T7-TRIM25ΔCC. 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. g Western blot analyses against T7 and DHX9 of pre-let-7a-1 pull-downs with HeLa TRIM25 KO cell extracts overexpressing T7-TRIM25ΔPRY/SPRY or T7-TRIM25-PRY/SPR. Lanes 1 and 4 represent 4% (40 μg) of the loading controls (input). Lanes 2 and 5 represent bead-only controls. Lanes 3 and 6 show pre-let-7a-1 pull-down