Structural Comparison of Pseudouridine-Modified U2 snRNA / Branchpoint Site Containing RNA Duplexes
W. J. Bauer, S. D. Kennedy, and C.L. Kielkopf
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry
Splicing of pre-mRNA is an essential process catalyzed by a spliceosome composed of >100 proteins and five small nuclear (sn)RNAs. A duplex between a consensus pre-mRNA site, called the branchpoint sequence (BPS), and a pseudouridine-modified region of the U2 snRNA contributes to positioning a conserved adenosine within the BPS into an extrahelical position for nucleophilic attack in the first catalytic step of pre-mRNA splicing. Based on previous NMR analysis of an RNA duplex containing S. cerevisiae U2 snRNA/BPS consensus sequences [Newby et al. (2002) NSMB 9:958], it was proposed that the U2 snRNA pseudouridine promotes the extrahelical conformation of a specific branchsite adenosine. However, our crystal structures of pseudouridine-modified U2 snRNA/BPS- containing RNA duplexes demonstrate the capacity of either the branchsite adenosine or a preceding purine nucleotide to assume a bulged conformation [Lin & Kielkopf (2007) Biochemistry 47:5503]. Although this latter observation is consistent with biochemical evidence that either of the two adjacent purines may shift into the bulged position [Query et al. (1994) Genes Dev. 8:587], the viewpoint of a pseudouridine-induced adenosine bulge has continued to dominate the field. By NMR analysis of (i) a duplex containing a pseudouridylated U2 snRNA and a mammalian BPS we here provide evidence for an intrahelical conformation of the 'branchsite' adenosine and a bulged conformation for the adjacent purine in solution. We further characterize the role of pseudouridines in branchsite selection by comparing this structure to BPS-containing duplexes with either (ii) an unmodified U2 snRNA or (iii) a U2 snRNA containing a second pseudouridine.