RNA hairpins with non-nucleotide spacers bind efficiently to the human U1A protein

The N-terminal RNA binding domain (RBD1) of the human U1A protein binds to the ten nucleotide loop in stemloop II of of U1 snRNA, and to its own 3' UTR structure. The nucleotides critical for recognition by the U1A RBD are displayed in very different geometric contexts in these two targets, leading to the question of what common features of RNA structure allow the RBD to efficiently recognize these two RNAs. The experiments described here used RNA hairpins, in which the loop size was altered by deletion, insertion or substitution with non-nucleotide (ethylene glycol)n spacers, to determine what features of this RNA structure were critical for interaction with the RBD1. Substitution of the three nucleotides on the 3' side of the RNA hairpin loop by (ethylene glycol)6-18 spacers does not significantly perturb the affinity, energetics or electrostatics of this RNA: protein association. These results confirm the suggestion that these loop nucleotides provide a flexible tether to allow the other seven nucleotides to fit onto the binding surface of the RBD, and lead to the hypothesis that conformational flexibility and the possible end-to-end distance of seven loop nucleotides are critical features of this complex formation.