Figure 4.

Purified recombinant Y1–3 proteins preferentially bind to an m⁶A-modified HIV-1 RNA fragment in vitro. A, structure of partial HIV-1 RNA 5′ UTR containing two GGACU motifs (gray background) that are labeled with words m⁶A #1 (nt 195–199) and m⁶A #2 (nt 239–243). PBS and DIS are highlighted in bold. The figure is adapted from Kharytonchyk et al. (30). B, sequences of synthesized control (Ctrl) RNA and m⁶A-modified (m⁶A RNA) fragment containing the second GGACU motif (bold) in HIV-1 RNA 5′ UTR corresponding to nt 235–281. The DIS-containing sequence (AAGCGCGC) was replaced with nucleotides GAG (underlined) to eliminate RNA dimerization. The A* indicates the m⁶A modification site in the RNA fragment. C, binding of Y1–3 proteins to m⁶A RNA versus control; unmodified RNA was monitored using the AlphaScreen assay. Data are from two independent experiments with biological triplicates. Results are shown as mean ± S.E. **, p < 0.005 by Mann-Whitney test. D, Y1–3 protein pulldown using biotin-labeled control and m⁶A RNA fragments. Streptavidin Dynabeads were used in the pulldown assay, and immunoblotting was performed using specific antibodies to Y1, Y2, or Y3. E, Y1–3 protein pulldown level based on densitometry analysis of the bands in immunoblotting. Data are average results from two independent experiments. F, calculated concentrations of Y1–3 proteins needed to reach 50% pulldown levels of 625 nm Y1–3 by the m⁶A RNA fragment.