Extended Data Fig. 10: TRIM71 CH-mutations lead to deregulation of Cdkn1a/p21 and Egr1 and loss of interaction to the NMD factor UPF1.
a,b) Representative immunoblots for the CLIP-qPCR assays in a) HEK293T cells and b) mESC. c-e) Protein levels of EGR1 and P21/CDKN1A in WT and mutant TRIM71 mESC. c) Representative immunoblots showing protein levels of EGR1 and P21/CDKN1A. d,e) Quantitation of EGR1 (d) and P21/CDKN1A (e) protein levels from immunoblots in (c). f-i) De-repression of CDKN1A 3′UTR in HEK293T cells overexpressing TRIM71 mutants and in Trim71 mutant mESCs. f) Schematic of luciferase assay to examine RNA target silencing by TRIM71 (CDKN1A as example). g) Luciferase reporter assay for the CDKN1A 3′UTR showing repression ability of the indicated TRIM71 constructs in HEK293T cells. Norm. RLU = normalized relative light units. h) Representative immunoblot showing TRIM71 construct overexpression for the CDKN1A-3′UTR luciferase assays in (g). i) Luciferase reporter assay for the CDKN1A 3′UTR showing repression ability of the indicated mESC line. j,k) CH-causing mutations impair TRIM71 binding to the NMD factor UPF1. Immunoblots show UPF1 enrichment upon co-precipitation with j) different Ig-tagged TRIM71 constructs transfected into HEK293T cells, namely Ig-Ctrl, Ig-TRIM71, Ig-ΔNHL6, Ig-R608H, and Ig-R796H or k) with endogenous FLAG-tagged TRIM71 and TRIM71-R595H in mESC. Statistical significance was tested by a two-sided, one-sample t-test (d, e, g, i): *: P<0.05, **: P<0.01, ***: P<0.001. Data represented as mean±s.e.m., overlaid with individual data points. For detailed statistical information (d, e, g, i), see Supplementary Table 13. Source data are provided.
