The structure of an essential splicing element: stem loop IIa from yeast U2 snRNA.

Abstract

Eukaryotic genes are usually transcribed as precursor mRNAs which are then spliced, removing introns to produce functional mRNAs. Splicing is performed by the spliceosome and provides an important level of post-translational control of gene expression. Stem loop IIa from U2 small nuclear (sn)RNA is required for the efficient association of the U2 small nuclear ribonuclear protein (snRNP) with the nascent spliceosome in yeast. Genetic analysis suggests that stem loop IIa is involved in RNA-protein interactions early in splicing, and it may also interact with other RNA sequences in U2. The sequence of loop IIa is well conserved, consistent with the idea that this loop is important for function. We have solved the structure of U2A, a 20-base analogue of stem loop IIa from Saccharomyces cerevisiae, using NMR and restrained molecular dynamics. In the process, we have demonstrated the efficacy of a new structure calculation protocol, torsion angle molecular dynamics. The structure that has emerged, which is consistent with the in vivo chemical protection data available for stem loop IIa in the context of intact U2 snRNA, contains a sheared GA pair followed by a U-turn in the loop. The U-turn conformation, which resembles the U-turns in tRNA anticodon loops, makes this stretch of U2 snRNA an obvious target for interactions with proteins and/or other RNA sequences. The phenotypes of many stem loop IIa mutants can be rationalized assuming that the U-turn conformation in the loop must be preserved for efficient splicing. This observation, combined with the phylogenetic conservation of its sequence, suggests that the conformation of the loop of stem loop IIa is essential for its function in pre-mRNA splicing.