The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

Abstract

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 59 end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents ''breathing'' of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 59 side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

Conformationally restricted nucleotides as a probe of structure–function relationships in RNA

“…6). This very high affinity complex (K D z 30 pM) has been examined structurally, biochemically, and kinetically (Scherly et al 1989(Scherly et al , 1990Lutz-Freyermuth et al 1990;Hall and Stump 1992;Hall 1994;Oubridge et al 1994;Hall 1998, 1999;Katsamba et al 2001Katsamba et al , 2002aLaw et al 2005Law et al , 2006bShowalter and Hall 2005). The basis for this tight interaction is an induced fit between regions of the protein and residues 1-7 of the unstructured 10-nucleotide (nt) loop, resulting in an intricate network of RNA-protein contacts (Hall 1994;Oubridge et al 1994;Hall 1998, 1999;Katsamba et al 2001;Pitici et al 2002;Law et al 2006a;Kormos et al 2007).…”

“…Binding experiments were performed on a BIACORE 2000 instrument (GE Healthcare) as described with small modifications (Katsamba et al 2002b;Law et al 2006b). RNA was diluted to a final concentration of 0.1 mM in HBS buffer (10 mM HEPES at pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) followed by heating at 80°C for 10 min and cooling to room temperature for 60 min to allow annealing of the stem.…”

“…RNA densities (including the wild-type reference channel) coated on the C5 chip were slightly higher than on the A6 surfaces to compensate for the weak binding of C5 LNA. Recombinant, C-terminally hexahistidine-tagged U1A protein (amino acids 1-101) (Law et al 2006b) was serially diluted in running buffer to the concentrations indicated in Figure 7 and injected at 20°C at a flow rate of 50 mL min À1 for 3 min (association phase). Disruption of any complex that remained bound after a 5-min dissociation was achieved using a 1-min injection of 2 M NaCl at 20 mL min À1 .…”

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

“…22,23 This points to the dominant roles of specific interactions in electrostatic rate enhancement. Experimentally, Law et al 15 studied the effect of RNA net charge on the binding rate by extending the U1SLII stem, which points away from U1A (Figure 1), and found only a very modest increase in k a .…”

“…The complex between the spliceosomal protein U1A and its target on the U1 small nuclear RNA has served as a model system for many studies. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In this complex, the N-terminal RNA recognition motif of U1A interacts in a sequence-specific manner with stem/loop II (U1SLII) of the U1 small nuclear RNA ( Figure 1). Both experimental and computational studies have probed determinants of the binding affinity, and a number of experimental studies 1,7,9,10,[13][14][15] have been carried out to dissect the binding rate.…”