(A–B) Paramagnetic relaxation enhancement (PRE) data from a spin label attached to a 4-thio-U nucleotide near the 3´-end of the ISS-N1 for (A) UP1-R140A protein bound to the modified ISS-N1 and (B) UP1-WT bound to the same modified ISS-N1. Secondary structure elements are drawn above the histograms. (C) Schematic representation of the modified ISS-N1 binding to hnRNP A1 RRMs. RRM2 is in red, and RRM1 in blue. The RNA sequence is written from the 5´-end to the 3´-end. The spin label is attached to a 4-thio-U located just after the RRM1 binding motif (AAGG). The spin label is represented as a yellow dot, and the PRE effect symbolized with a faint halo in yellow. (D) Surface representation of the residues in UP1-WT affected by the presence of the spin label near the 3´-end of the modified ISS-N1 RNA. The surface of hnRNP A1 RRMs is represented in red and blue for RRM2 and RRM1, respectively. Residues for which PRE data is not available due to a missing assignment in the bound form or due to severe signal overlaps are colored in gray in order to make them not appear as ‘not affected’. Residues with a ratio of the intensity in the oxidized or paramagnetic state over the intensity in the reduced or diamagnetic state (Ipara/Idia) lower than 0.7 are colored in yellow. For facilitating the structural interpretation of the PRE data, the positions of the O4 atoms of the U6 residue within the NMR bundle of hnRNP A1 RRM1 bound to 5´-UUAGGUC-3´ (this study) are shown as pink spheres. The spin label attached to the 4-thio-U in the modified ISS-N1 should sample the space around this approximate position. (top) front view. (bottom) 180 ° rotation, back view. (E) Schematic representation of hnRNP A1 RRMs binding to the modified ISS-N1-u25g RNA. RRM2 is in red, and RRM1 in blue. RRM2 binds the 5´ motif and accommodates three nucleotides (CAG), with the AG dinucleotide being stacked onto F108 and F150 of the RNP2 and RNP1 motifs, respectively. RRM1 binds the 3´ motif and accommodates four nucleotides (AAGG), with the central AG dinucleotide being stacked onto F17 and F59 of the RNP2 and RNP1 motifs, respectively. (F) Structural model of the ISS-N1-u25g bound to UP1-R140A. Modeling was performed as described in the Appendix 2. RRM2 is in red, RRM1 in blue, and the inter-RRM linker in green. The ISS-N1-u25g RNA is in yellow. Some nucleotides are labeled in order to appreciate the path of the RNA on the RRMs. Please note that the path of the RNA-spacer between the 5´ and 3´ motifs is not restrained by experimental constraints. The present structural model therefore illustrates one possible path of the ISS-N1 on the RRMs and should not be seen as the unique conformation adopted by the RNA spacer.