The stem cell–specific protein TRIM71 inhibits maturation and activity of the prodifferentiation miRNA let-7 via two independent molecular mechanisms

TRIM71 interferes with the last processing step of let-7 biogenesis in ESCs. (A) Representation of relative expression differences in the miRNome of wild-type (WT, Trim71^fl/fl) and Trim71 knockout (KO, Trim71^−/−) ESCs (GSE62509). MiRNAs were classified as previously described into four categories (gonad-specific, brain-specific, ESC-specific, or somatic miRNAs) and displayed as percentages (n = 590). (B) Scatter plot showing the relative fold change (FC, with −1 ≤ FC > +1) of differentially expressed miRNAs in Trim71 KO cells plotted against their baseline expression level in WT ESCs (GSE62509). Guide strands (5p) of all let-7 family members are marked. (C) RT-qPCR showing relative expression of the indicated miRNAs in WT and Trim71 KO ESCs (n = 3–10). (D) RT-qPCR showing relative expression of let-7a, (E) let-7g and (F) miR-294 miRNAs in WT ESCs transiently overexpressing GFP-Ctrl or GFP-TRIM71 48 hpt (n = 3). (G) RT-qPCR showing relative pri-, pre- and mature let-7a expression in WT and Trim71 KO ESCs (n = 3–5). (H) RT-qPCR showing relative guide (5p) and (I) passenger (3p*) strand levels of mature let-7a and let-7g miRNAs in in WT and Trim71 KO ESCs (n = 3–5). RT-qPCR quantification of all miRNAs was normalized to the levels of the housekeeping U6 snRNA. Error bars represent SD. (***) P < 0.005, (**) P < 0.01, (*) P < 0.05, (ns) nonsignificant (unpaired Student's t-test). See also Supplemental Figures 1, 2.