Book of Abstracts: Albany 2003
June 17-21 2003
Role of Pseudouridine in Stabilizing the Pre-mRNA Branch Site Helix of the Eukaryotic Splicosome
Excision, or splicing, of noncoding regions (introns) from precursor (pre)-mRNA in eukaryotes is catalyzed by the spliceosome, a ribonucleoprotein complex comprising recyclable small nuclear (sn)RNA and protein components. An early step in assembly of the catalytic core involves pairing of a consensus sequence of the intron with a short complementary region of the U2 snRNA to form a short helix with a single unpaired adenine base. The 2'OH of this adenosine, called the branch site, is the nucleophile in the cleavage reaction at the pre-mRNA 5? splice site. A pseudouridine (ψ) residue in the region of U2 snRNA that opposes the 5' neighbor of the branch site adenosine has been identified in all eukaryotes investigated to date. Using NMR spectroscopy, we have determined high resolution structures of ψ-modified and unmodified duplexes representing the pre-mRNA branch site of S. Cerevisiae.
The structure of the unmodified branch site duplex displays a continuous A-type helical geometry, with the branch site base stacked within the helix. A markedly different structure is seen for the ψ-modified duplex (figure below). The backbone of the intron strand in the branch site region has a pronounced kink at the level of the branch site adenosine (A24 in the figure), extruding the base from the helix, which forms a nearly coplanar base triple the A-U base pair two positions upstream. The ψ-modified structure is consistent with all previous biochemical and genetic data. The chain reversal also exposes the 2'OH of the branch site adenosine to the widened major groove of the helix, which provides a structural basis for recognition and access to the nucleophile by the RNA substrate strand in the first step of splicing.
We then asked how ψ, which differs from U primarily in the presence of an additional NH group, favors formation of such a markedly different structure. The orientation and dimensions of the major groove in the vicinity of ψNH1 (?extra? imino group of ψ) in the branch site structure could accommodate a water molecule. NMR investigation of cross relaxation between the ψNH1 and water are consistent the model of a water-mediated hydrogen bond involving ψ as the source of added stabilization or structural change.
The U2 strand of ψBP has a sharp chain reversal at A24, the branch site adenosine, extruding the A24 base from the helix. The nine lowest energy structures consistent with NMR-derived data converged, with a pairwise RMSD of 1.36 Å (all atoms; 1.30 &Aing; for heavy atoms). The extrahelical base is stabilized by a base triple (upper); chain reversal at A24 results in exposure of its 2?OH (lower).
Nancy L. Greenbaum*
Dept. of Chemistry & Biochemistry and
Institute of Molecular Biophysics