TRIM71 depends on LIN28A for the down-regulation of let-7 expression in ESCs. (A) Schematic representation for the generation of the indicated ESC lines. Lin28a KO ESCs were generated from WT (Trim71fl/fl) ESCs via TALENs. Trim71 KO and Trim71–Lin28a double KO were generated by addition of 4-OHT to WT and Lin28a KO ESCs, respectively (see Materials and Methods for details). (B) RT-qPCR showing relative mRNA expression of Trim71 and (C) Lin28a in the generated ESC lines (n = 6–8). (D) RT-qPCR showing relative expression of pri-let-7a, (E) pre-let-7a, (F) mature let-7a-5p and (G) mature let-7g-5p miRNAs in the different ESC lines (n = 4–8). (H) Let-7 reporter assay after transient transfection of a Renilla luciferase reporter under the control of a 3′UTR containing 8x Let-7 binding sites (BS) in the different ESC lines (n = 3). (Norm. RLU) Normalized relative light units. (I) RT-qPCR showing relative expression of mature let-7a-5p and (J) Trim71 mRNA in WT and Lin28a KO ESCs transiently overexpressing FLAG-Ctrl or FLAG-TRIM71 48 hpt (n = 3–6). RT-qPCR quantification of miRNAs and mRNAs was normalized to the levels of the housekeeping U6 snRNA and Hprt mRNA, respectively. Error bars represent SD. (***) P < 0.005, (**) P < 0.01, (*) P < 0.05, (ns) nonsignificant (unpaired Student's t-test between WT and each KO condition, unless indicated by a line joining the two compared conditions). (K) Immunoblot showing the RNA-independent coprecipitation of endogenous TRIM71 with Ig-tagged LIN28A overexpressed in wild-type ESCs. PABP was used as a control for an RNA-dependent interaction with LIN28A. (L) Immunoblot showing the coprecipitation of endogenous LIN28A with endogenous TRIM71 in wild-type ESCs. See also Supplemental Figures 3, 4.
