Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise

Abstract

U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein-protein interaction modules. UHM-ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM-ULM modules for protein-protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome.

Keywords: RRM; SF3B1; U2AF1; UHM; protein–protein interaction.

FIGURE 1.

Multiple UHM–ULM interactions guide the early stages of spliceosome assembly and other pathways. Experimentally confirmed phosphorylation sites of the ULM-containing regions are marked by pink circles. “W” represents ULM tryptophans. Thick gray arrow represents the transition during spliceosome assembly. Thin black arrows represent known UHM–ULM interactions. Arrows toward SF3b155 indicate the general ULM-containing region rather than specific binding sites.

FIGURE 2.

UHM–ULM recognition. (A) Schematic representation of consensus UHM (cyan)–ULM (green) interactions. (B–F) Structures of UHM–ULM complexes, including (B) U2AF⁶⁵ UHM–SF1 ULM (PDB ID: 4FXW); (C) U2AF³⁵ UHM–U2AF⁶⁵ ULM (PDB ID: 1JMT); (D) U2AF²³ UHM–U2AF⁵⁹ ULM (PDB ID: 4YH8); (E) SPF45 UHM–SF3b155 ULM5 (PDB ID 2PEH); and (F) CAPERα UHM–SF3b155 ULM5 (PDB ID: 4OZ1). Magenta spheres mark phosphorylation sites: the SF1 and SF3b155 sites in B,E,F have been confirmed experimentally, whereas the human U2AF⁶⁵ site in C is the predicted phosphorylation of a TP motif. The surface of the central ULM tryptophan is colored yellow, UHM RXF-motifs are blue, and acidic UHM α-helical residues are red; orange spheres are zinc ions on the back surface of the U2AF²³ UHM in D.

FIGURE 3.

RNP interactions. (A) N-terminal RRM of U1A prototype bound to RNA ligand. The RNP surfaces of UHM (cyan)–ULM (green) complexes, including (B) U2AF⁶⁵ UHM–SF1 ULM (PDB ID: 4FXW); (C) U2AF²³ UHM–U2AF⁵⁹ ULM (PDB ID: 4YH8); (D) SPF45 UHM–SF3b155 ULM5 (PDB ID 2PEH); and (E) CAPERα UHM–SF3b155 ULM5 (PDB ID: 4OZ1). The conserved aromatic residue at RNP1 position 5 typically is masked by a C-terminal extension (magenta) of the core UHM fold. The key residues at positions 2 of RNP2 and positions 3/5 of RNP1 are colored blue. Orange spheres represent zinc ions. Indigo, space-filling residues in C correspond to MDS-relevant mutations of the human homolog. Views are following an ∼120° rotation about the y-axis relative to Figure 2.