Role of the central junction in folding topology of the protein-free human U2-U6 snRNA complex analyzed by time-resolved FRET
Intron removal during splicing of precursor pre-mRNA requires assembly of spliceosomal small nuclear (sn)RNAs into catalytically competent conformations to promote two transesterification reactions. U2 and U6 snRNA are the only snRNAs directly implicated in pre-mRNA splicing catalysis, but rearrangement and remodeling steps prior to catalysis require numerous proteins. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations characterized by four and three helices surrounding a central junction in equilibrium. To analyze the role of the central junction in positioning the two helices critical for formation of the active site, we used ensemble time-resolved fluorescence resonance energy transfer to measure distances between fluorophores at selected locations in constructs representing the protein-free human U2-U6 snRNA complex. Data describing four angles in the four-helix conformer suggest the complex adopts a tetrahedral geometry; addition of Mg2+ results in shortening of the distances between neighboring helices, indicating compaction of the complex around the junction. In contrast, the three-helix conformer shows a closer approach between the two helices bearing critical elements, but addition of Mg2+ widens the distance between these stems. Presence of Mg2+ also enhances the steady state fraction of the three-helix conformer found to be active in spliceosomes. Although the central junction assumes a significant role in orienting helices, in neither conformer, with or without Mg2+, are the critical helices positioned sufficiently close to favor interaction, implying that a major role of spliceosomal remodeling proteins is to overcome such distances to create and stabilize a catalytically active fold.