Figure 1: UNR binds to roX2 directly.

(a) Schematic representation of roX2 and derivatives. The stem–loops (SL), the roX boxes and the limits of roX2 A–G fragments are indicated. (b) Analysis of UNR binding to roX2 fragments by EMSA. (c) Analysis of UNR binding to SLs in the 3′ half of roX2 by RNA pulldown. Three copies of MS2 coat protein-binding sites (MS2) fused to the RNA of interest are recognized by MBP-tagged MS2 coat protein, which in turn binds to amylose beads. After adding recombinant UNR, RNPs are washed and eluted. Quantifications correspond to the average of UNR binding in three experiments. Full-size blots corresponding to this Figure are shown in Supplementary Fig. 5. (d) Footprinting of UNR on full-length roX2 treated with RNase T2. cDNAs obtained by primer extension were electrophoretically separated. Lanes labelled U, G, C, A represent Sanger sequencing ladders. Nucleotides are numbered relative to nucleotide +1 of roX2. Protected nucleotides are labelled with open circles (black, yellow and red for no, weak and strong protections, respectively) and nucleotides that gain accessibility upon UNR binding are indicated with filled circles. The region of SL7 potentially remodelled by UNR is indicated by a red square. A schematic summary of the experimental footprinting data is shown on the right. The insets show independent experiments at higher resolution, or different exposures of the same gel.