The interaction between TRIM71 and LIN28 is mediated by the uridylating enzyme TUT4. (A) Representative immunoblot showing the coprecipitation of endogenous TUT4 with ectopically expressed Ig-TRIM71 in control HEK293T cells (siCtrl)—which lack LIN28A expression—and upon LIN28B knockdown (siLIN28B). (B) Representative immunoblot showing the coprecipitation of ectopically expressed FLAG-TRIM71 with endogenous LIN28B in control (siCtrl) and TUT4 knockdown (siTUT4) HEK293T cells. (C) Representative immunoblot showing the RNA-independent interaction between TRIM71 and TUT4 or LIN28B. AGO2 was used as a control of an RNA-dependent interaction. (D) Representative immunoblot showing the coprecipitation of endogenous TUT4 with different Ig-tagged TRIM71 constructs in HEK293T cells, depicted in F. (E) Representative immunoblot showing the coprecipitation of endogenous TUT4 with Ig-tagged TRIM71 and C12LC15A overexpressed in HEK293T cells. (F) Schematic representation of TRIM71 constructs used for IP assays in D and E. For each construct, present domains are depicted in black, deleted domains are depicted in gray and mutations are marked with a white “x.” See also Supplemental Figure 8.
