TRIM71 relies on AGO2 binding for the specific repression of let-7 activity. (A) Let-7 reporter assay upon overexpression of FLAG-Ctrl or FLAG-TRIM71 in control (siCtrl), LIN28B knockdown (siLIN28B) and TUT4 knockdown (siTUT4) HEK293T cells. Ectopic let-7 miRNA duplex expression was used as a control for efficient let-7 reporter repression, but revealed a regulation of mature let-7 activity (n = 4–6). (B) TRIM71-mediated let-7 activity repression strength, calculated from values in A, as Norm. RLU (FLAG-TRIM71)/Norm. RLU (FLAG-Ctrl) for each condition. (C) Let-7 reporter assay in HEK293T cells upon FLAG-Ctrl, FLAG-TRIM71 or FLAG-LIN28A coexpression with either a control miRNA duplex (miR Ctrl), a let-7a precursor stem–loop (pre-let-7a), or a mature let-7a duplex (let-7a) (n = 3–8). (D) Let-7 reporter assay after overexpression of FLAG-Ctrl or FLAG-TRIM71 in wild-type (WT) and AGO2 knockout (KO) HEK293T cells (n = 6–8). (E) TRIM71-mediated let-7 activity repression strength, calculated from values in D, as Norm. RLU (FLAG-TRIM71)/Norm. RLU (FLAG-Ctrl) for each condition. (F) Schematic representation of TRIM71 constructs used in G and H. For each construct, present domains are depicted in black, deleted domains are depicted in gray and mutations are marked with a white “x.” (G) Let-7 reporter assay in HEK293T cells upon overexpression of different FLAG-tagged TRIM71 constructs depicted in F (n = 3–10). (H) Representative immunoblot showing the coprecipitation of endogenous AGO2 with different Ig-tagged TRIM71 constructs—depicted in F—overexpressed in HEK293T cells. (I) Representative confocal microscopy images showing the partial colocalization of GFP-tagged TRIM71 and AGO2 in perinuclear foci. (Norm. RLU) Normalized relative light units. Error bars represent SD. (***) P < 0.005, (**) P < 0.01, (ns) nonsignificant (unpaired Student's t-test between the main control condition and each other condition, unless indicated by a line joining the two compared conditions). See also Supplemental Figure 10.
