A novel protein-protein interaction in the RES (REtention and Splicing) complex

Abstract

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions.

Keywords: Gene Regulation; Protein Complex; Protein Structure; Protein-protein Interaction; RNA-binding Protein; Spliceosome; U2AF Homology Motif.

FIGURE 1.

Sequence and structural features of RRM and UHM domains. a, structure-based sequence alignment of UHM/RRM domains. Secondary elements are indicated on the top and residue numbers of Snu17p are given at the bottom. Residues in the RNP motifs of Snu17p and Arg-X-Phe motifs of UHM domains are colored cyan and red, respectively. Conserved residues in the motifs are highlighted by black boxes. b, sequence alignment of ULM peptide sequences known to bind UHM domains with the Bud13p ligand (residue numbers of Bud13p indicated at the bottom). The invariant tryptophan of ULM ligands and the two glycine residues that are unique to Bud13p are colored orange and magenta, respectively. Residues that form the binding interface between Snu17p and Bud13p are highlighted with a lime green background in a and b, respectively. c and d, structures of a canonical RRM domain (PDB code 2G4B) in complex with RNA (c) and a representative UHM domain (PDB 2PEH) bound to a ULM peptide (d). RNP motifs and the UHM-specific RXF sequence are annotated in β strands.

FIGURE 2.

Mutational analyses of the Bud13p-Snu17p interaction. a, interactions between Snu17p, Bud13p, and Pml1p were assayed by one-step Ni-NTA purification of wild type or mutant proteins co-expressed in E. coli. Supernatant (S) and pellet (P) fractions from bacterial lysates as well as flow-through (F) and elution (E1, E2, and E3) fractions from the Ni-NTA purifications are shown after Coomassie staining of the gels. M indicates the molecular mass marker with size indicated on the left in kDa. Note that Pml1p is less well stained than Snu17p and Bud13p. b, complementation of Δbud13 strains with an empty vector, or plasmids expressing wild type or mutant Bud13p was monitored through the growth of serial dilutions of transformants on selective plates at the indicated temperatures. c, Western blot showing the level of wild type and mutant Bud13p is detected through the fused TAP tag. d, complementation of a Δsnu17 strain was performed as for bud13 in b. e, the levels of wild type and mutant Snu17p were monitored by detection of the TAP tag by Western blotting.

FIGURE 3.

ITC and NMR analysis of Snu17p binding to Bud13p. a, four different Bud13p peptides containing the invariant Trp residue were tested by ITC titrations for Snu17p binding. Binding curves for a 66-mer, a 35-mer used in the accompanying structural studies, a 22-mer, and a 14-mer Bud13p peptide, are depicted from left to right. Dissociation constants obtained by fitting the isotherms after subtracting the heat of dilution are listed. b, overlay of ¹H,¹⁵N HSQC spectra of free Snu17p (residues 1–113, black) and after titration with the shortest Bud13p ligand (residues 225–238, orange) or with the optimal Bud13p peptide (residues 222–256, green). c, secondary ¹³C chemical shifts Δδ(¹³C^α)-Δδ(¹³C^β) for Snu17p free and bound to Bud13p-(222–256), {¹H}-¹⁵N heteronuclear NOE of the Snu17p-Bud13p complex, and chemical shift perturbations of Snu17p amide signals (recorded on a sample of ¹⁵N-labeled Snu17 residues 25–113) upon binding to Bud13p-(222–256) are plotted versus the amino acid sequence of Snu17p. Secondary structural elements of Snu17p are indicated on top.

FIGURE 4.

NMR analysis of Bud13p ligand (residues 222–256) binding to Snu17p. a, overlay of the ¹H,¹⁵N HSQC spectra of the Bud13p ligand free and when bound to Snu17p. b, secondary ¹³C chemical shifts Δδ(¹³C^α-¹³C^β) and {¹H}-¹⁵N heteronuclear NOE data for the Bud13p ligand when bound to Snu17p. The last 5 residues of Bud13p have negative NOE values and are not shown. The short helix in the C-terminal region of the peptide is indicated on top.

FIGURE 5.

¹H (ω₃),¹H (ω₁) planes from ¹³C/¹⁵N-filtered, ¹³C-edited NOESY-HSQC spectra in the binary complex composed of doubly labeled Snu17p and unlabeled Bud13p. The ¹³C chemical shifts (ppm) of the selected ω₂ planes are indicated on top. The experiment selects NOE cross-peaks between protons bound to ¹²C or ¹⁴N in Bud13p and protons bound to ¹³C in Snu17p, providing exclusively intermolecular restraints. For each cross-peak the upper and lower atom name refers to Snu17p and Bud13p assignments, respectively.

FIGURE 6.

Structure of the Snu17p-Bud13p complex. a, overlay of the 20 lowest energy structures of Snu17p (blue) in complex with Bud13p (yellow). The N and C terminus of each protein is annotated. b, schematic representation of the Snu17p-Bud13p complex with amide CSP induced by Bud13p binding mapped onto the structure with a color gradient from white to blue with increasing CSP. The invariant Trp residue of Bud13p is shown as stick representation. c, the RNP signature motifs of Snu17p (Tyr³⁴, Phe⁷⁴, Tyr⁷⁶) are conserved but not entirely exposed to the solvent. d, electrostatic surface of Snu17p with negative and positive charges colored red and blue, respectively.

FIGURE 7.

Details of the intermolecular contacts in the Snu17p-Bud13p complex. a, the Bud13p peptide forms an arch around the helical binding surface of Snu17p. b, close-up views of the interaction at the entry point (left), the U-turn (middle), and the exit point (right) of the Bud13p peptide shown in a. Interacting residues are annotated and colored blue for Snu17p and yellow for Bud13p. Intermolecular hydrogen bonds to the Bud13p backbone are indicated by dashed lines.

FIGURE 8.

Comparison of ligand binding modes of the UHM/RRM-fold. In all panels the UHM/RRM domains are shown in the same orientation. a, three examples of the canonical UHM-ULM interaction. b, PTB binding to Raver1 ligands, which lack a Trp residue. c, two different binding modes of peptide partners with a Trp residue by ALYREF protein. d, the novel Bud13p peptide recognition by Snu17p.