Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45

AUF1 p45 and AUF1 p45^aDMA support the annealing of the UAR and CS cyclization sequences of the WNV genome. (A) Scheme of the fluorescence-based assay to detect an RNA annealing activity of AUF1 p45 or AUF1 p45^aDMA. Annealing of the complementary WNV 5′- and 3′UAR and WNV 5′- and 3′CS RNAs that are fluorescently labeled with Cy5 (5′UAR, 5′CS) and Cy3 (3′UAR, 3′CS), respectively, leads to a detectable FRET signal. (B, left panel) The assay (5′UAR–3′UAR RNA annealing assay) was performed with 10 nM of Cy5-labeled 5′UAR RNA that was incubated with the indicated concentrations of recombinant AUF1 p45 and AUF1 p45^aDMA, respectively. Following the addition of 10 nM Cy3-labeled 3′UAR RNA, the fluorescence emission of the Cy5 fluorophore was measured for 400 sec. The fluorescence signals were plotted as a function of time and fitted according to a second-order reaction (Equation 3; see Materials and Methods). (Right panel) The observed rate constants k_obs (sec⁻¹) were plotted as a function of the concentration of AUF1 p45 and AUF1 p45^aDMA; they are given in Supplemental Table S5. (C) Same as in B except that the complementary 5′- and 3′CS sequences of the WNV genome were used. The measured k_obs (sec⁻¹) values of the 5′CS–3′CS RNA annealing assay are given in Supplemental Table S6.