A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pair ing between the U2 small nuclear (sn)RNA and a complementary consensus sequ ence of the intron, called the branch site, results in positioning of the 2 'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes r epresenting the paired U2 snRNA and intron sequences from Saccharomyces cer evisiae were examined by solution NMR spectroscopy. Oligomers were synthesi zed with pseudouridine (psi) at a conserved site on the U2 snRNA strand (op posite an A-A dinucleotide on the intron strand, one of which forms the bra nch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviat ion from helical parameters and an extrahelical orientation for the unpaire d adenosine. Incorporation of psi also stabilized the branch-site interacti on, contributing -0.7 kcal/mol to duplex DeltaG(37)degrees. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and im ply that the extrahelical orientation of the branch-site adenosine may faci litate recognition of this base during splicing.