Fig. 6: The role of the UPF1-activator, UPF2, in context of histone mRNA decay.

A Comparison of the unwinding activity of UPF1-CHh on SL RNA substrate in the absence (gray trace) and presence of UPF2s (blue trace). The unwinding activity of UPF1-Hel is shown as a reference (black trace). Data presentation and statistical analysis for this experiment and Fig. 6B are as described for Fig. 5A, B. The inset shows the first 10 min of the reaction, where the differences in unwinding activity are the largest. The significance of the differences in unwinding observed in this experiment and in Fig. 6B as well as p-values are indicated. B Unwinding activity of UPF1-CHh on the SL RNA substrate in the presence of either UPF2 (blue trace) or SLBPfl (red trace) or both UPF2 and SLBP (purple trace). The activity of UPF1-CHh in absence of UPF2 and SLBPfl (black trace) has been shown for comparison. The inset shows an enlarged view of the first 10 min of a 60-min reaction. Activation of UPF1 by UPF2 reduces the inhibitory effect of SLBP on the helicase. C Schematic representation of the domain organization of human UPF2. The structured middle-of-eIF4G (MIF4G) domains are shown as filled rectangles while the C-terminal U1-binding domain (U1BD) is depicted by a crosshatch. Variants used in this study are shown below. D GST-pulldown assay of GST-UPF2s (and GST-GYF as negative control) with full-length SLBP and 3′hExo. 3′hExofl shows a specific interaction with UPF2s while SLBPfl shows no appreciable binding. E GST-pulldown assays using GST-3′hExofl as a bait and different UPF2 variants as prey show that all UPF2 proteins encompassing the acidic linker (AL) are co-precipitated by 3′hExo. F Analytical SEC of mixture of UPF2-AL and 3′hExo (black trace) confirms that the UPF2-AL is sufficient for formation of a stable complex with 3′hExo. SEC runs of the individual proteins 3′hExofl (green trace) and UPF2-AL (blue trace)1`123 are shown for comparison. The corresponding SDS-PAGE analysis are shown below. See also Supplementary Fig. 6A. The experiment was performed independently three times with similar results.