Solution structure of the U2 snRNP protein Rds3p reveals a knotted zinc-finger motif

Abstract

Rds3p, a component of the U2 snRNP subcomplex SF3b, is essential for pre-mRNA splicing and is extremely well conserved in all eukaryotic species. We report here the solution structure of Rds3p, which reveals an unusual knotted fold unrelated to previously known knotted proteins. Rds3p has a triangular shape with a GATA-like zinc finger at each vertex. Pairs of cysteines contributing to each finger are arranged nonsequentially in a permuted arrangement reminiscent of domain-swapping but which here involves segments of subdomains within a single chain. We suggest that the structure arose through a process of segment swapping after gene duplication. The fingers are connected through beta-strands and loops, forming an overall topology strongly resembling a "triquetra knot." The conservation and surface properties of Rds3p suggest that it functions as a platform for protein assembly within the multiprotein SF3b complex of U2 snRNP. The recombinant protein used for structure determination is biologically active, as it restores splicing activity in a yeast splicing extract depleted of native Rds3p.

Fig. 1.

NMR ensemble and backbone representations of Rds3p. (A) Stereoview of overlay of deposited ensemble of 20 lowest-energy models of Rds3p. The N and C termini, which are at the rear of the structure as viewed, are marked, as are clusters 1, 2 and 3 (the zinc atom of cluster 1 is obscured in this view). Significant disorder is limited to the region adjacent to cluster 3 (residues 51–55; see Overall Structure of Rds3p) on the far right of the structure as shown. The structure is oriented with the β-triangle facing upward. (B) Corresponding stereoview of the lowest energy structure from the NMR ensemble shown in chainbow coloring as a backbone ribbon with cysteine sidechains.

Fig. 2.

Sequence alignment and overall structure of Rds3p. (A) Sequence alignment of human and yeast Rds3p. Components of the structure are shown above the sequence, using the same color code as in 2C (K, zinc knuckle; β, β-hairpin; S, strand; H, helix; L, loop; numbers denote fingers 1, 2, and 3). (B) Ribbon stereoview of the Rds3p structure in chainbow coloring (corresponding to the rainbow color gradient bar in 2A) with the β-triangle facing downwards (180° rotation from Fig. 1). Sidechains of the metal-binding cysteine residues are shown numbered. (C) Schematic showing how Rds3p is built from three GATA-like zinc fingers (shown in same orientation as B). In Rds3p, each β-hairpin is broken and replaced by a strand (magenta) running from one finger to the next, and a loop (orange) is added from the α-helix of one finger to the zinc-knuckle of the next. The zinc knuckle of finger 3 is “interrupted.” (D) Simplest representation of the origin of the knot in the Rds3p structure (same orientation as in B and C).

Fig. 3.

Sequence of the ordered part of Rds3p showing the nonsequential arrangement of the three zinc clusters.

Fig. 4.

Hydrophobic core of Rds3p. (A) The strands connecting the three zinc fingers form a β-triangle and are highly stabilized by hydrogen bonds. (B) Conserved hydrophobic residues between the face of the zinc fingers and the β-triangle stabilize the core of Rds3p.

Fig. 5.

CD spectra of Rds3p at different temperatures and in vitro splicing assays. (A) Overlay of far UV CD spectra of Rds3p at 20°C (light blue) after heating to 95°C (dark blue) and after recooling the sample to 20°C (pink). The spectra recorded at 20°C almost perfectly overlap, indicating that Rds3p recovers its original structure after this heating cycle. (B) In vitro splicing assay with TAP-SF3b and recombinant Rds3p. GAL1::rds3-1 strain grown under splicing supporting conditions shows that activity is not affected by addition of TAP-purified SF3b complex or recombinant Rds3p. (C) Time course of splicing activity in an extract of GAL1::rds3-1 strain grown in the presence of glucose at 37°C. No splicing activity can be observed in this extract unless it is supplemented with TAP-purified SF3b complex (as a control) or recombinant Rds3p.

Fig. 6.

Electrostatic surface of Rds3p. Regions of positive charge are shown in blue, and regions of negative charge are shown in red. (Left) The bottom view (same orientation as in Fig. 1) shows a basic patch formed by lysine and arginine residues on strand 2 at the base of the triangle. (Right) The top view (same orientation as in Fig. 2 B and C) shows a slightly acidic patch, but otherwise the surface is relatively featureless. The missing area in the top view results from truncation of the flexible tails.