Structures of SF3b1 reveal a dynamic Achilles heel of spliceosome assembly: Implications for cancer-associated abnormalities and drug discovery

Abstract

The pre-mRNA splicing factor SF3b1 exhibits recurrent mutations among hematologic malignancies and cancers, and consequently is a major therapeutic target of clinically-advanced spliceosome inhibitors. In this review, we highlight and rigorously analyze emerging views of SF3b1 conformational transitions, including the human SF3b particle either in isolation or bound to spliceosome inhibitors, and human or yeast spliceosome assemblies. Among spliceosome states characterized to date, an SF3b1 α-helical superhelix significantly closes to surround a U2 small nuclear RNA duplex with the pre-mRNA branch point sequence. The SF3b1 torus is locally unwound at an active site adenosine, whereas protein cofactors appear to stabilize overall closure in the spliceosome. Network analyses demonstrates that the natural SF3b1 dynamics mimic its conformational change in the spliceosome, raising the possibility of conformational selection underpinning spliceosome assembly. These dynamic SF3b1 conformations have consequences for gatekeeping of spliceosome assembly and therapeutic targeting of its cancer-associated dysfunction.

Keywords: HEAT repeat; Hsh155; Myelodysplastic syndrome; Pre-mRNA splicing, cancer; Ribonucleoprotein structure.

Figure 1.

Diagram of SF3b1-containing complexes during spliceosome assembly.

Figure 2.

(A) Domain organization of SF3b1. Recurrent cancer-associated mutations are expanded above (yellow highlight). Amino acids for which substitutions confer pladienolide-resistance are named (green font). HR, dihelical HEAT repeat; ULM, U2AF Ligand Motif. (B) Structure of the SF3b particle (PDB ID 5IFE). (C) Representative structure of the B^ACT spliceosome (PDB ID 5Z58 shown to illustrate intron position). SF3b1, cyan; U2 snRNA, violet; pre-mRNA, magenta. Mutational hotspots are yellow spheres; pladienolide-resistance sites are green spheres; branch point adenosine (BP-A) is shown as hot pink spheres.

Figure 3.

(A) SF3b1 interactions with the BPS – U2 snRNA duplex. The PHF5a subunit is omitted for clarity and would overlay the branch point adenosine (BP-A, magenta surface) facing the viewer. Sites of pladienolide-resistant mutations are green. (B) SF3b1 interactions with the pre-mRNA intron. PDB ID 5Z58. Mutational hotspots are colored yellow. For reference between panels, the pre-mRNA nucleotides are numbered relative to the BP-A. HR, HEAT repeat.

Figure 4.

(A) Comparison of human SF3b1 conformation in the isolated SF3b particle (yellow, PDB ID 5IFE) with the B^ACT spliceosome (cyan, PBD ID: 6FF4, a representative structure of the human B^ACT at relatively high resolution, Table 1) following alignment of the C-terminal repeat (residues 1244-1285). (B) View rotated 90° about the x-axis relative to (A). Results are similar following comparison of SF3b1 of the SF3b particle with other spliceosome structures. A movie portraying the conformational transition is given as supplementary information (Supplementary Movie S1).

Figure 5.

Internal pocket of the SF3b1 torus in the (A) SF3b particle (yellow, PDB ID 5IFE) or (B) B^ACT spliceosome (cyan, PDB ID 6FF4) calculated using the Castp server (http://sts.bioe.uic.edu/castp/) with a 1.8 Å probe radius.

Figure 6.

Relationship between consecutive HEAT repeats of the SF3b1 superhelix in the context of the isolated particle (black, PDB ID 5IFE) or spliceosome (teal, PDB ID 6FF4). The plots analyze the intramolecular step from one HEAT repeat to the next in the indicated superhelix. The steps are numbered on the x-axis for clarity: Step 1 corresponds to the relationship of HEAT repeat 1 (HR1) to HR2, Step 2 corresponds to the relationship of HR2 to HR3, and so forth. (A) The rotational angle (twist) between the principal axes of α-helices from consecutive HEAT repeats. (B) Bar graph of the differences in the rotational angle of consecutive HEAT repeats between SF3b1 in the two contexts. The average values and standard deviation for the two relatively high resolution human B^ACT structures (PDB ID’s 6FF4 and 5Z58) compared to the SF3b particle are given. Unpaired, two-tailed t-tests with Welch’s correction analyze the rotational difference of each repeat relative to the average difference over all repeats: ***, p <0.0005; **, p <0.005; *, p <0.05; not significant, p >0.05. Repeats with significant differences are colored teal. (C) Translational shift (pitch) between centroids of α-helices from consecutive HEAT repeats. No significant differences in pitch of the superhelix are observed among structures. Results from comparison of the C-terminal α-helix of each di-helical HEAT repeat are shown (residue ranges in Table S1), which avoids a disordered region of SF3b1 in the isolated particle (residues 1093 – 1106). Similar results are obtained from comparison of the intact dihelical units as well as human or yeast homologues (Fig. S1). Representative scripts are given as Supplementary Materials.

Figure 7.

(A) The distances between the centroids of the HEAT repeats following alignment of the human SF3b1 structures in the SF3b particle (PDB ID: 5IFE) compared to B^ACT spliceosome (PDB ID: 6FF4). (B-C), Anisotropic network model analysis (ANM 2.0, http://anm.csb.pitt.edu/) shows natural modes of SF3b1 motion similar to its conformational changes for spliceosome integration. The core HEAT domain (residues 463-1274) is analyzed. (B), Plot of the major normalized modes (fluctuations, a measure of flexibility) per SF3b1 residue. The directions of mode 1 (red) and mode 2 (blue) respectively close and twist the SF3b1 torus. (C), Heat map of normalized fluctuations in mode 1, in a color gradient from blue for no movement to orange for maximum movement. Mode 2 appears similar, although the direction of movement differs.

Figure 8.

(A) Comparison of the SF3b1 conformation in the SF3b particle (gray, PDB ID 5IFE) compared to SF3b bound to inhibitors pladienolide B (PB) (cyan, PDB ID 6EN4) or E7107 (magenta, PDB ID 5ZYA). PB is shown as yellow spheres for reference. (B) Close view of SF3b1 interactions with PB (yellow surface) (PDB ID 6EN4). SF3b1 residues for which mutations confer pladienolide-resistance are green. PHF5a is shown in gray. The interactions of SF3b1 with E7107 (PDB ID 5ZYA) are nearly identical with the exception of an additional cycloheptyl-piperazine moiety.