Fig. 3: RRM4 binding is crucial for complex stabilization and translation enhancement.

a Removal of the pyrimidine stretch in LinkEF (same mutation as shown in (b) for the Lmut Luciferase Reporter Assay construct) interferes with RNA binding of RRM4 and broadens the distance distribution measured between spin labels attached to RRM1 and RRM34 significantly. Compared to free PTBP1, the distance distribution narrows upon binding of EMCV-IRES-DtoF, but the mean distance does not shift substantially. This effect is less pronounced with the Lmut. Semi-transparent areas in distance distribution plots correspond to 95% confidence intervals. b Constructs for the Luciferase Reporter Assay (top) with the deleted or mutated (pyrimidine to purine) sequence of EMCV-IRES-DtoF (bottom, only one mutation at a time). c Luciferase Reporter Assay given as IRES-mediated translation efficiency with respect to the WT-IRES. Remarkably, the removal of the RRM4-binding site (Lmut) results in the same activity loss as the deletion of the whole EMCV-IRES-DtoF sequence, which links the importance of RRM4 binding for complex stabilization and rigidification with translational efficiency of the IRES. The error bars represent the standard deviations of the three biological replicates. The value for each biological replicate was determined as a mean of three technical replicates. The data underlying panels (a, c) are provided as Source Data.