TRIM71 represses let-7 expression and activity via two independent mechanisms. (A) Representative immunoblot showing the levels of endogenously expressed TRIM71 and LIN28 proteins in the indicated cell lines, used for experiments depicted in B–E. (B) RT-qPCR showing the basal expression levels of mature let-7a miRNA in the indicated cell lines (n = 3 6). (C) Let-7 reporter assay upon transient transfection of a Renilla luciferase reporter under the control of a 3′UTR containing 8x Let-7 binding sites (BS) in the indicated cell lines (n = 3). (Norm. RLU) Normalized relative light units. (D) RT-qPCR showing the expression of mature let-7a miRNA in the indicated cell lines upon overexpression of FLAG-tagged TRIM71, C12L15A and LIN28A (n = 3–8). (E) Let-7 reporter assays in the indicated cell lines upon overexpression of FLAG-tagged TRIM71, C12L15A, and LIN28A (n = 3–8). (Norm. RLU) Normalized relative light units. RT-qPCR quantification of let-7a was normalized to the levels of the housekeeping U6 snRNA. Error bars represent SD. (***) P < 0.005, (*) P < 0.05 (unpaired Student's t-test between FLAG-Ctrl and each other condition). See also Supplemental Figure 9.
