Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

Abstract

SUMMARY The essential splicing factors U2AF65 and SF1 cooperatively bind consensus sequences at the 3′ end of introns. Phosphorylation of SF1 on a highly conserved ‘SPSP’ motif enhances its interaction with U2AF65 and the pre-mRNA. Here we reveal that phosphorylation-induces essential conformational changes in SF1 and in the SF1/U2AF65/3′ splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF65 at 2.29 Å resolution, and of the unphosphorylated SF1 domain at 2.48 Å resolution, demonstrate that phosphorylation induces a disorder-to-order transition within a novel SF1/U2AF65 interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full length (P)SF1/U2AF65/3′ splice site assembly. We further determine that SPSP phosphorylation and the novel SF1/U2AF65 interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.

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

“…As discussed below, a structure of the U2AF 65 UHM bound to the N-terminal domain of SF1 further revealed that regions beyond the minimal ULM stabilize this masked UHM conformation ( Fig. 3B; Wang et al 2013;Zhang et al 2013). It is possible that analogous, ligand-mediated reinforcements of the α-helical block on the UHM "RNA binding surface" will emerge as more UHM-containing structures are determined bound to intact, ULM-containing proteins.…”

“…Phosphorylation of this site by cGMP-dependent protein kinase-I reduces SF1 association with U2AF 65 and thereby inhibits spliceosome assembly. Structurally, the phosphorylated SF1 S20 would disrupt a hydrogen bond and electrostatically repel a conserved aspartate residue (D401) in the acidic α-helix of the U2AF 65 UHM (Wang et al 2013). Two of the SF3b155 ULMs display serine residues at analogous positions as SF1 S20, including SF3b155 S216 and S336 in ULM2 and ULM5; phosphorylation of SF3b155 S216 has been confirmed in an acute myeloid leukemia cell line by mass spectrometry (Weber et al 2012).…”

“…1B, 2B; Wang et al 2013;Zhang et al 2013). The N-terminal α-helix of the SF1 coiled-coil mediates the primary contacts between this domain and the U2AF 65 UHM (Fig.…”

“…This SPSP-phosphorylation event subtly increases the binding affinity of SF1 for U2AF 65 (Manceau et al 2006;Zhang et al 2013), most likely by stabilizing the coiled-coil fold of the phosphorylated SF1 domain. Structures of the SPSP-phosphorylated SF1 bound to the U2AF 65 UHM shows that the phosphorylated serines coordinate three arginine residues and induce folding of an otherwise disordered linker between the two α-helices of the coiled-coil (Wang et al 2013;Zhang et al 2013). The UHM-containing KIS kinase specifically phosphorylates the SPSP motif of SF1 (Manceau et al 2006), and KIS-knockout mice showed subtle changes in gene expression and specific behaviors, including spontaneous activity and contextual fear conditioning (Manceau et al 2012).…”

“…New structures of larger U2AF and SF1 complexes implicate regions adjoining the ULMs as accessories for UHM engagement (Wang et al 2013;Zhang et al 2013;Yoshida et al 2015). Phosphorylation sites within and flanking the ULMs can regulate the marriage of phosphorylated SF1 with the U2AF 65 UHM (Wang et al 2013;Zhang et al 2013) or exacerbate the divorce of the phosphorylated ATX1 ULM from the U2AF 65 UHM in lieu of a 14-3-3 binding partner (Lim et al 2008;de Chiara et al 2009). Still questionable are the potential polygamous versus monogamous relationships of the multiple SF3b155 ULMs with different UHM-binding partners.…”