Fig. 2: ITC and CLIR-MS/MS analysis of RNA binding by TRIM25 CC domain.

a Representative ITC binding isotherm for TRIM25 CC:pre-let-7 complex (n = 3). The value shown in the figure is the average of all replicates and its standard deviation. b The surface potential representation of the CC dimer (PDB:4LTB) indicates a positively charged surface. The enlargement of this interface shows potential RNA binding residues. Mutation of residues K283 and K285, shown in grey, leads to a TRIM25-CC mutant we term m2. In purple, the peptide detected by CLIR-MS/MS (see below) is highlighted including the four potential RNA binding residues. Mutation of these four residues leads to a TRIM25-CC mutant we term m4. The structure shown was generated using UCSF ChimeraX⁸⁰. c Representative ITC binding isotherm for TRIM25 CC-m2 with pre-let-7 complex (n = 3). d Schematic representation of the CLIR-MS/MS method. The RNA binding protein (grey) is crosslinked to an equimolar mixture of unlabelled (black) and uniformly ¹³C/¹⁵N-labelled RNA (red). e MS spectrum of the TRIM25-derived peptide GISTKPVYIPEVELNHK crosslinked to a single U nucleotide (see methods). f Representative ITC binding isotherm for TRIM25 CC-m4:pre-let-7 complex (n = 3). All experimental setups and ITC measurements including replicates are listed in Supplementary Table 1.