UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

Abstract

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional coactivator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophancontaining peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

U2 AF 65 assemblies drive sequence‐specific splice site recognition

“…U2AF 65 reinforced the recruitment of CAPERα to GST‐SF3b155c but not that of PUF60 and SPF45 (Fig A). A similar effect was observed when adding a high‐affinity complex of U2AF 65 with a minimal domain of U2AF 35 that should not allow the access of CAPERα to the ULM of U2AF 65 (Fig A, right panel and Appendix Fig S3) . U2AF 65 addition did not lead to an efficient pulldown of CAPERα with GST‐SF1c (GST‐SF1_1‐255; Fig EV1A).…”

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

“…Indeed, UHM-containing proteins exhibit binding preferences for distinct ULM-containing proteins (Table 3). The U2AF 35 UHM-U2AF 65 ULM complex is more than 1000-fold preferred over offtarget U2AF 35 UHM-SF1 ULM or CAPERα-U2AF 65 ULM interactions (Corsini et al 2007;Stepanyuk et al 2016). Likewise, the U2AF 65 UHM-SF1 ULM complex is at least 200-fold preferred over an erroneous U2AF 65 UHM-U2AF 65 ULM homodimer (Corsini et al 2007).…”

Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells

“…16 A paralogue of U2AF65, RBM39, is involved in the pre-mRNA splicing pathways in humans and Drosophila. 17,18 U2AF1 (also named U2AF35) is the U2 small nuclear RNA cofactor, which includes zinc finger domain, UHM and RS domain structures.…”