Solution structure determination of the spliceosomal U2snRNA - intron helix: Role of a conserved pseudouridine in branch site conformation

Maintenance of the integrity of the genetic information encoded in eukaryotic DNA is dependent upon an efficient and extremely accurate splicing mechanism. The process by which introns are spliced from precursor messenger (pre-m)RNA molecules and exons are ligated together is a fundamental process in gene expression that is carried out by the spliceosome, a macromolecular machine comprised of recycleable small nuclear (sn)RNA and protein components. Specific RNA-RNA recognition events in the spliceosome regulate the two-step transesterification reaction required for intron removal and exon ligation, and a catalytic core predominantly composed of RNA forms an active site for this chemistry. One essential element of the spliceosomal catalytic core is a short helix formed by the pairing of the U2 snRNA with a consensus sequence of the intron. This pairing features a single unpaired adenosine residue, called the branch site, the 2'OH of which is responsible for initiating nucleophilic attack at the 5' splice site phosphate during the first step of splicing. These studies investigate the structural role of a conserved pseudouridine (psi) residue in U2 snRNA juxtaposed from the branch site adenosine. The solution NMR structures of two duplex constructs representing the U2 snRNA and intron sequences from S. Cerevisiae, one containing a pseudouridine at the conserved site and one containing a uridine in place of the modified base, were solved using homonuclear methods. These structures show that the presence of the psi residue in the branch site helix induces a different conformation for the branch site adenosine as compared with its unmodified counterpart. The psi-induced structure explains recognition of the branch site base by splicing factors and places the nucleophile in an accessible position for the first step of splicing. Subsequent ROESY-type experiments suggest that the structural basis for the branch site conformational switch facilitated by psi is its ability to form a water-mediated hydrogen bond in the major groove of the helix, stabilizing this alternative branch site architecture.