Methylation of AUF1 p45 increases the binding affinity of the protein to the WNV RNA termini and leads to an enhanced stimulation of WNV RNA synthesis. (A) Schematic drawing of the organization of the applied RNA transcripts. The longest transcript, the WNV sgRNA, consists of the 5′ and 3′ UTR and a part of the core coding sequence; accordingly, it contains the 5′ SLA, the 3′ SL, all cyclization elements, and the AU-rich element in the upstream portion of the 3′ UTR (indicated as differently shaded boxes). The organization of the other transcripts follows this scheme; the sequence of the unrelated RNA that was mostly used in these experiments is given in Supplemental Table S3. (Column 3) The binding affinities of AUF1 p45 and AUF1 p45aDMA with the different RNAs were determined by filter binding (see Materials and Methods). Fitting the curves according to a cooperative binding revealed the apparent binding constant Kapp. (Column 2) The Kapp values with AUF1 p45 were obtained by reevaluation of binding curves that were published earlier (Friedrich et al. 2014). The results and standard deviations from at least three different experiments are shown. (n.d.) Not determined. (B) 200 nM of nonmethylated AUF1 p45 and methylated AUF1 p45aDMA were tested by supplementation in the in vitro replicase assay with sgRNA template. Newly synthesized [32P]-labeled RNA products were analyzed on a denaturing Tris-Borate PAGE by phosphorimaging. The levels of induction of RNA synthesis are shown in comparison to the control (replicase assay in the absence of AUF1 p45; set to 1). Results of three independent experiments are shown; error bars reflect standard deviations.
