Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition

Abstract

Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex.

Figure 1.

Proteins and domains involved in 3′-splice site recognition. (A) Diagram of the SF1–U2AF65 3′-splice site recognition complex representing domains of SF1 and U2AF65 (HH, helix hairpin; KH, hnRNP K homology; QUA2, quaking homology-2; RRM, RNA recognition motif; ULM, U2AF homology domain (UHM) ligand motif). SF1 and U2AF65 are coloured blue and green, respectively, with the same colour code for the proteins being used in the NMR spectra throughout the article. (B) Diagram of the protein constructs used. (C) Sequence alignment of SF1^HH domains. Sequences were taken from the UniProt database (www.uniprot.org) and residue numbers are given for Homo sapiens SF1. The secondary structure of SF1^HH is depicted above the alignment. The phosphorylated serine residues (Ser80 and Ser82) are indicated in red. Sequences were aligned with ClustalW (18) and analysed with Jalview 2 (19).

Figure 2.

Structure of a novel helix hairpin in the N-terminal domain of SF1. (A) Ribbon representation of the ensemble of the 10 lowest energy structures and surface representation coloured according to electrostatic surface potential at 3 kT/e⁻ for positive (blue) or negative (red) charge potential using the program APBS (36). Close-up view of the N-terminal (B) and C-terminal (C) structured extensions. Side chains of key residues mediating the interactions between the α-helices and the N/C termini are shown as sticks.

Figure 3.

NMR analysis of the SF1^NTD–U2AF65^UHM interaction. (A) Superposition of ¹H,¹⁵N HSQC NMR spectra of labelled SF1^NTD and U2AF65^UHM free (black) and when bound to unlabelled U2AF65^UHM (blue) or SF1^NTD (green), respectively. Selected residues, which are shifted upon formation of the SF1^NTD–U2AF65^UHM complex are annotated. (B) CSPs of amides linked to U2AF65^UHM (blue) and SF1^NTD (green) binding. To selectively analyse the contributions of the secondary binding interface, the CSPs obtained in a titration of labelled SF1^ULM with unlabelled U2AF65^UHM were subtracted from the SF1^NTD CSPs. Residues with strong CSPs that are therefore located in the secondary binding interface are annotated.

Figure 4.

Structure of the SF1^NTD and U2AF65^UHM complex. (A) Ribbon representation of the ensemble of the 10 lowest energy structures and surface representation coloured according to electrostatic surface potential at 3 kT/e for positive (blue) or negative (red) charge potential using the program APBS (36). (B) Close-up view of SF1^NTD–U2AF65^UHM complex interface. Side chains of key residues mediating the interactions between the α-helices and the N/C-termini are shown as sticks.

Figure 5.

NMR and SAXS analysis of the effect of tandem serine phosphorylation of SF1. (A) SDS–PAGE analysis of phosphorylation of SF1^NTD and SF1. The phosphorylated protein (red arrow) migrates slower on the gel than the non-phosphorylated. (B) Superposition of ¹H,¹⁵N HSQC NMR spectra of non-phosphorylated (black) and phosphorylated SF1^NTD and SF1 (red). Selected residues, which are shifted upon phosphorylation are annotated. (C) CSPs (red) and residue-specific local correlation times τ_c are shown for SF1^NTD (blue) and pSF1^NTD (red). The secondary structures and the phosphorylation sites are depicted above the diagram. The tandem phosphorylated linker region that rigidifies upon phosphorylation is highlighted by a box. A ribbon representation of SF1^HH colour coded with the phosphorylation-induced CSPs is shown. (D) SAXS data showing comparisons of radial density distributions of non-phosphorylated and phosphorylated SF1^NTD and SF1, in complex with U2AF65^UHM, U2AF65^RRM123 and with U2AF65^RRM123–RNA, respectively.

Figure 6.

Cooperative binding of U2AF65^RRM123 and SF1 to a 3′-splice site RNA. RNA was incubated with buffer or U2AF65^RRM123 (0.2, 0.5, 1 and 2 µM; indicated by triangles) in the absence or presence of 1.5 µM His₆-tagged SF1^2-320 or internal deletion mutants (A) or with 6.6 µM SF1 or pSF1 (B). Reaction products were separated by native PAGE and visualized by autoradiography. The migration of SF1–U2AF65–RNA complexes (closed arrowhead) and SF1–RNA complexes (open arrowhead) is indicated. (C) Role of SF1^HH and the SF1^NTD–U2AF65^UHM interaction in the formation of the 3′-splice site recognition complex. SF1^HH may establish an optimal orientation of the SF1 (KH–QUA2) and U2AF65^RRM1,2 RNA-binding subunits in the complex and thereby support cooperative RNA binding. Tandem phosphorylation of SF1 might contribute to the formation of the ternary complex by stabilizing a yet unknown interface.