Role of the central junction in folding topology of the protein-free human U2-U6 snRNA complex.

Huong Chu, William Perea, Nancy L Greenbaum

RNA (New York, N.Y.) 2020 Jul

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U2 and U6 small nuclear (sn)RNAs are the only snRNAs directly implicated in catalyzing the splicing of pre-mRNA, but assembly and rearrangement steps prior to catalysis require numerous proteins. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations in equilibrium, characterized by four and three helices surrounding a central junction. The four-helix conformer is strongly favored in the in vitro protein-free state, but the three-helix conformer predominates in spliceosomes. To analyze the role of the central junction in positioning elements forming the active site, we derived three-dimensional models of the two conformations from distances measured between fluorophores at selected locations in constructs representing the protein-free human U2-U6 snRNA complex by time-resolved fluorescence resonance energy transfer. Data describing four angles in the four-helix conformer suggest 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 helices bearing critical elements, but the addition of Mg2+ widens the distance between them; thus in neither conformer are the critical helices positioned to favor the proposed triplex interaction. The presence of Mg2+ also enhances the fraction of the three-helix conformer, as does incubation with the Prp19-related protein RBM22, which has been implicated in the remodeling of the U2-U6 snRNA complex to render it catalytically active. These data suggest that although the central junction assumes a significant role in orienting helices, spliceosomal proteins and Mg2+ facilitate formation of the catalytically active conformer. © 2020 Chu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Citation

Huong Chu, William Perea, Nancy L Greenbaum. Role of the central junction in folding topology of the protein-free human U2-U6 snRNA complex. RNA (New York, N.Y.). 2020 Jul;26(7):836-850