The methylated AUF1 p45 shows an enhanced RNA chaperone activity. (A) Interactions of 3′ Cyc RNA and 5′ UTR RNA tested as a mobility shift on a native PAGE. The interactions were monitored in the absence and presence of AUF1 p45 and AUF1 p45aDMA, respectively. Two nanomoles of 3′ Cyc RNA was exposed to the indicated amounts of recombinant AUF1 p45 or AUF1 p45aDMA, followed by the addition of 2 nM [32P]-labeled 5′ UTR RNA; the added protein was removed before the analysis by PAGE. (Left) A representative gel is shown; the migration positions of the unbound 5′ UTR RNA and of the 5′–3′ RNA complex are indicated. (Right) The levels of 5′–3′ RNA complex formation were quantified and evaluated for statistical significance (RNA complex formation in the absence of protein set to 1); (*) P ≤ 0.05; (**) P ≤ 0.01. (B, top) Scheme of the fluorescence-based assay (3′SLtrunc–5′UAR interaction assay) to detect AUF1 p45-mediated conformational RNA rearrangement by dequenching of Cy5 (see text). (Bottom) The assay was performed with 10 nM of Cy5-labeled 3′ SLtrunc that had been incubated with the indicated concentrations of recombinant AUF1 p45aDMA. Following the addition of 100 nM 5′ UAR, the fluorescence emission was measured for 400 sec. The fluorescence signals were plotted as a function of time and fitted according to a first-order reaction (without protein; Equation 1) or second-order reaction (in the presence of protein; Equation 2). Note that no increase of fluorescence emission was detectable if the reaction was performed in the absence of 5′ UAR. (C) The observed rate constants kobs (sec−1) were plotted as a function of the protein concentration (see Supplemental Table S4). The kobs values for AUF1 p45 and hnRNPH1 were taken from Friedrich et al. (2014).
