Fig 2: Crystal structure of the DHX15-SUGP1 G-patch complex. (A) Schematic drawing of the structure of the human DHX15-SUGP1 G-patch complex. The domains in DHX15 are given different colors and labeled. The SUGP1 G-patch is in magenta. The bound adenosine diphosphate (ADP) molecule is shown as a stick model (gray for carbon atoms) and Mg2+ ion as a sphere (pink). WH, winged helix; OB, oligonucleotide/oligosaccharide binding. (B) The structure of DHX15 is shown as a molecular surface, colored by domains. The SUGP1 G-patch interacts with the WH and RecA2 domains. (C) Overlay of the structures of human DHX15-SUGP1 G-patch complex (in color) and DHX15-NKRF G-patch (DHX15 in gray and NKRF in orange). (D) Interaction of the N-terminal part of the SUGP1 G-patch with the DHX15 WH domain. DHX15 is shown as a surface, and side chains in the SUGP1 G-patch (in magenta) making large contributions to the interface are shown as sticks. The NKRF G-patch is shown for comparison (orange). The Cα atoms of glycine residues are indicated by spheres. DHX15 residues that were selected for mutation (SI Appendix, Fig. S1) are colored in green and labeled in dark gray. (E) Interaction of the C-terminal part of the SUGP1 G-patch with the DHX15 RecA2 domain. (F) Crystal structure of the C-terminal region of SUGP1 (aa 433–577). The four helices are labeled, and the side chains of Met569 and Met573 are shown as sticks. (G) A dimer of the C-terminal region of SUGP1 in the crystal. The second monomer is shown in cyan, and the twofold axis of the dimer is vertical. Conformational differences for helix αA in the two monomers are visible. The structure figures are produced with PyMOL (www.pymol.org).

Fig 3: Expression of several DHX15 mutants partially recapitulates the splicing defects of mutant SF3B1. (A) HEK293T cells were transfected with Vec or expression plasmid for HA-tagged SF3B1 (WT or K700E) or HA-tagged DHX15 (WT or one of the mutants as indicated), followed by 32P RT-PCR of the cryptic 3′ss (open arrowheads) and canonical 3′ss (solid arrowheads) of the indicated genes. (B) Western blotting of protein extracts from HEK293T cells in (A). anti-HA, rabbit polyclonal anti-HA tag antibody. (C) The ORAI2 minigene or its mutant (–38 A > G) minigene was co-transfected with Vec or expression plasmid for HA-tagged SF3B1 (WT or K700E) or HA-tagged DHX15 (WT or one of the mutants as indicated), followed by 32P RT-PCR of the cryptic 3′ss (open arrowheads) and canonical 3′ss (solid arrowheads) from the ORAI2 minigenes.

Fig 4: DHX15 specifically associates with the SUGP1 G-patch. (A, Upper) Schematic representation of plasmid constructs (1 to 4) expressing FLAG-GST tandem tags, FLAG-GST-tagged SUGP1 (aa 543–645), its G574A-G582A mutant, and FLAG-GST-tagged SUGP1 (aa 607–645), respectively. (Lower) Each of the four constructs was transfected into HEK293T cells, followed by small-scale affinity purification using FLAG and GST tags. The purified proteins in eluates were resolved by SDS-PAGE, followed by silver staining. (B) HEK293T cells were transfected with construct 2 in (A), followed by large-scale affinity purification using FLAG and GST tags. The purified proteins were resolved by SDS-PAGE, followed by staining with QC Colloidal Coomassie Stain (Bio-Rad). (C) Aliquots of the purified proteins in (A) were resolved by SDS-PAGE, followed by western blotting. In all panels: M, Precision Plus Protein marker (Bio-Rad).

Fig 5: Mutations in the DHX15-SUGP1 G-patch fusion protein abolish the rescue effect. (A, Upper) Schematic representation of plasmid constructs (1 to 5) expressing HA-tagged DHX15-SUGP1 (aa 543–645) fusion protein and its mutants P449E, P533E, R222G, and D260N, respectively. (Lower) Vec or expression plasmid for HA-tagged SF3B1 (WT or K700E) was co-transfected with Vec or one of the five constructs (as indicated) in HEK293T cells, followed by 32P RT-PCR of the cryptic 3′ss (open arrowheads) and canonical 3′ss (solid arrowheads) of the indicated genes. (B) Quantification of the 32P RT-PCR products in (A). PSI of the cryptic 3′ss of the indicated genes is shown. Error bars represent SDs of the means (n = 3; three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired, two-tailed, and unequal variance t test). (C) Western blotting of protein extracts from HEK293T cells in (A).