Structural Investigation of Mutated Spliceosomal U2 SnRNA — Intron Helices

The removal of non-coding sequences, or introns, from precursor messenger (m)RNAs and the ligation of coding sequences, or exons, a process called pre-mRNA splicing, is an essential step in gene expression. The spliceosome is the biomolecular machinery responsible for catalysis of splicing in eukaryotic nuclei; it comprises five small nuclear (sn)RNA and many protein components, which participate in a dynamic assembly of RNA-protein and RNA-RNA interactions. Of major importance is the RNA-RNA pairing between segments of the U2 snRNA and intron to be spliced, called the branch site helix, which positions the 2'-hydroxyl group of a single unpaired adenosine (A) to act as the nucleophile for the first of two transesterification reactions. The solution NMR structure of a short duplex representing the branch site helix showed that a pseudouridine (ψ, a naturally occurring modified base) in a phylogenetically conserved position of U2 snRNA is a key element in inducing the branch site A to adopt an extrahelical conformation. This conformation was shown to be stabilized by formation of a base triple involving the branch site A and a highly conserved purine-pyrimidine base pair. Conservation of RNA sequences in this region suggests that this motif is important for the chemistry of splicing and/or recognition processes during spliceosome assembly. To determine the importance of base triple formation in the observed branch site conformation, spectroscopic studies were performed on native and mutated RNA sequences designed to preserve or discourage base triple formation, respectively. Intensity of 2-aminopurine (2AP) fluorescence was used to determine the relative orientation of the branch site A in different constructs. 2AP fluorescence intensity of ψ-modified duplexes that contained either an AU or GC base pair (i.e. the native purine-pyrimidine orientation) emitted relatively high fluorescence intensity, suggesting solvent exposure and extrahelical position of the branch site A. In contrast, 2AP fluorescence intensity in a mutated construct with a reversed orientation of the AU base pair (i.e. AU→UA) was substantially lower, consistent with an intrahelical (stacked) position for the branch site A in the mutated construct. In no case was the A 5' to the branch site residue extruded from the helix. Structural features from NMR studies of the mutated ψ-modified duplex (i.e. AU→UA) showed an intrahelical conformation for the bulged region as compared to native constructs, which exhibited features of an extrahelical branch site A. By comparison with chemical shifts of the native ψ-modified duplex, resonances of exchangeable and non-exchangeable protons of bases were upfield shifted and cross-strand NOEs were observed. The results corroborate those data from 2AP fluorescence data, suggesting intrahelical stacking of the branch site A derived in the absence of base triple formation. Data from UV melting studies were consistent with the structural features deduced from other spectroscopic methods. These findings support a model where by the base triple formed in the ψ-dependent branch site sequences of the U2 snRNA-intron helix maintains a role in recognition of the branch site A by other splicing factors and/or in stabilizing the nucleophile prior to the first step of splicing.