Complex role of the β2-β3 loop in the interaction of U1A with U1 hairpin II RNA

Abstract

RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a β-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting β-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the β2-β3 loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the β2-β3 loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys50 and Met51, which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys50 and Met51 with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys50 or Met51 caused a dramatic loss in stability of the U1A-U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the β2-β3 loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA-protein complex.