Figure 3. Titration of ISS-N1 variants to UP1 R140A variant.
Overlays of 2D (15N,1H)-HSQC spectra of the free UP1 R140A variant (blue) and UP1 R140A in the presence of 1 equivalent of ISS-N1 variants (red). The region shown in a close-up is shown on the respective panel (E–H). Residues pointed out for RRM1 in the text are marked in panels (F) and (H). NMR signals can be obtained at a good linewidth for the complex of UP1 R140A + ISS-N1-u25g. (A) UP1 R140A (0.3 mM) + ISS-N1 WT. (B) UP1 R140A (0.3 mM) + ISS-N1-14g25g. (C) UP1 R140A (0.3 mM) + ISS-N1-c14g. (D) UP1 R140A (0.3 mM) + ISS-N1-u25g. (E–H) Close-up view of the region boxed on panels A–D).
Figure 3—figure supplement 1. Sequence and localization of the cis-acting element ISS-N1 in the SMN2 intron 7.
(A) Sequence of the region around the ISS-N1 in the intron 7 of SMN2 (Hua et al., 2008; Singh et al., 2006). The ISS-N1 is boxed in red, with the two AG dinucleotides constituting the core of the hnRNP A1 binding sites in italic. Exonic sequences are in upper case, whereas intronic sequences are in lower case. (B) ISS-N1 oligonucleotides wild-type and variants (c14g, u25g and 14g25g) used in NMR studies. Residue numbering such as used in this manuscript corresponds to the residue number from the 5´ splice-site and is present above the sequences. The 5´ GGA sequence that allows high in vitro transcription efficiency is in gray. The two AG dinucleotides constituting the core of the hnRNP A1 binding sites are in bold. They are separated by a spacer of 9 nucleotides.
Figure 3—figure supplement 2. Titration of ISS-N1 variants to UP1 wild-type.
Overlays of 2D (15N,1H)-HSQC spectra of the free UP1 (blue) and UP1 in the presence of 1 equivalent of ISS-N1 variants (red). The region shown in a close-up is marked by a box. (A) UP1 WT (0.4 mM) + ISS-N1 WT. (B) UP1 WT (0.2 mM) + ISS-N1-c14g. (C) UP1 WT (0.2 mM) + ISS-N1-u25g. (D) UP1 WT (0.2 mM) + ISS-N1-14g25g.