TRIM71 interacts with LIN28 proteins and specifically regulates let-7 miRNAs in HEK293T cells. (A) Schematic representation of both LIN28 paralog proteins (LIN28A and LIN28B) domain organization. (CSD) Cold shock domain, (ZKD) zinc knuckle domain, (NoLS) putative nucleolar localization sequence, (NLS) nuclear localization signal. (B) Representative immunoblot showing GFP-tagged TRIM71 and C12LC15A coprecipitation with endogenous LIN28B in HEK293T cells. (C) Representative immunoblot showing GFP-tagged TRIM71 and C12LC15A coprecipitation with Ig-tagged LIN28A overexpressed in HEK293T cells. Overexpression of an Ig-empty control vector together with wild-type GFP-TRIM71 (first lane) excluded an Ig-mediated TRIM71–LIN28A interaction. (D) Representative immunoblot showing ectopic TRIM71 and endogenous LIN28B protein levels in HEK293 cells stably overexpressing GFP-Ctrl or GFP-TRIM71. (E) RT-qPCR showing relative expression of LIN28B mRNA (n = 3) and (F) the indicated mature miRNAs (n = 6) in HEK293 cells stably overexpressing GFP-Ctrl or GFP-TRIM71. RT-qPCR quantification of miRNAs and mRNAs was normalized to the levels of the housekeeping U6 snRNA and HPRT1 mRNA, respectively. Error bars represent SD. (***) P < 0.005, (*) P < 0.05, (ns) nonsignificant (unpaired Student's t-test). See also Supplemental Figure 7.
