Fig. 2.
Tandem dsRBDs of Stau1 form a composite binding platform for UPF2. a Schematic representation of the Stau1 constructs designed to map the UPF2-binding site. The orange rectangle labeled “DD” represents the dimerization domain of the NF-κB transcription factor p5033, connected to Stau1 via a 4-residue linker. The native dimerization module of Stau1, comprising the SSM and dsRBD5 domains, is denoted by “dd”. b, c GST-pulldown assays of the Stau1 constructs with GST-UPF2s as a bait. Negative controls using GST are shown in Supplementary Fig. 2a, b. The dsRBD3 domain of Stau1 is necessary for binding UPF2, although a strong interaction between UPF2 and Stau1 requires the presence of at least two such binding-competent dsRBDs. Contaminants that co-purified with GST-UPF2 are indicated by asterisks (*). d Analytical size-exclusion chromatography (SEC) depicting stable complex formation between UPF1, UPF2s, and a Stau1 construct containing only the UPF2-binding site (dsRBD2-3-DD). The terms Abs and Vr denote the absorbance and retention volume of the proteins in this and all subsequent chromatograms. The exclusion volume of the column is 0.8 mL. The corresponding SDS-PAGE analysis of the peak fractions (indicated on the chromatogram) is shown on the right. Formation of a stable Stau1-UPF complex is mediated by UPF2. The source data for this figure are provided as a source data file