SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

Abstract

Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3' splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.

Keywords: HEAT motif; Prp5; SF3B1/Hsh155; disease mutation; pre-mRNA splicing fidelity.

Figure 1.

SF3B1 mutations found in human diseases cause altered branch region splicing fidelity in Saccharomyces cerevisiae. (A) Seven conserved residues of human SF3B1 that are frequently mutated in human MDS and CLL diseases (Yoshida et al. 2011; Quesada et al. 2012) and their counterparts in S. cerevisiae Hsh155p. (B) Scheme of ACT1-CUP1 splicing reporters. Mutant reporters used in this study are indicated in red; the BS–U2 RNA duplex is indicated. (C) ACT1-CUP1 copper reporter assays indicate that most disease mutation-containing hsh155 alleles either improved or exacerbated splicing activities of the BS-U257C reporter but not of the wild-type reporter. Multiple mutations at residues H331 and K335 were tested; other residues were based on human disease mutations. (D) Intron mutations that reduce pairing with U2 snRNA (U257C and A258C) are sensitive to hsh155 mutations analogous to those found in human disease, whereas the 5′SS, 3′SS, and branch nucleophile mutations are not.

Figure 2.

Screen for factors that improve splicing of the BS-U257C suboptimal BS–U2 duplex substrate. (A) Strategy for the UV mutagenesis screen of S. cerevisiae mutant alleles that improve splicing of the BS-U257C mutant reporter. Mutations were identified by a combination of traditional genetics and genome sequencing techniques (see the Results). (B) List of identified yeast alleles with enhanced splicing activity of BS-U257C. (C) Only intron mutations reducing the BS–U2 duplex stability (U257C and A258C) are sensitive to screened alleles, whereas others are not.

Figure 3.

Identification of interacting motifs between Hsh155p and Prp5p by in vitro protein interaction assays. (A) Schematic of domains/motifs in Hsh155p and Prp5p. (B) HEATs 1–8, 5–12, and 9–16 of Hsh155p are pulled down by Prp5p, whereas other regions are not. (C) HEATs 9–12 of Hsh155p are sufficient for binding to Prp5p. (D) The integrity of HEATs 1–6 is important for their interaction with Prp5p. (E) The N terminus of Prp5p is required for Hsh155p–Prp5p interaction. GST-tagged Hsh155p and 6xHis-tagged Prp5p were expressed and purified from E. coli. Pull-down assays were performed using Ni-NTA agarose beads, and GST alone was used as negative control.

Figure 4.

Genetic screen for hsh155 alleles that alter splicing of the BS-U257C reporter and potentially change interaction with Prp5p. (A) Schematic of a genetic screen for hsh155 alleles that were generated by error-prone PCR and improve splicing of BS-U257C. PCR products containing the mutated hsh155 region (N terminus to HEAT 11) were cotransfected with the linearized wild-type HSH155-LYS2 plasmid into a yeast strain carrying the HSH155 gene on URA plasmid followed by homologous recombination (gap repair). (B) Confirmation of 22 isolated single-residue mutated hsh155 alleles that improve splicing of U257C. (C) Ten hsh155 alleles selected for further analysis. Mutated sites in HEAT motifs are listed.

Figure 5.

hsh155 alleles that alter BS splicing fidelity exhibit altered interaction with Prp5p in vitro. (A) hsh155 alleles that improve splicing of BS mutant reporters in vivo (top) exhibit enhanced interaction with Prp5p in vitro (bottom). (B) hsh155 alleles that inhibit splicing of BS mutant reporters in vivo (top) exhibit inhibited interaction with Prp5p in vitro (bottom). (C) Prp5p-DPLD motif mutants that improve splicing of BS mutant reporters (Shao et al. 2012) exhibit enhanced interaction with Hsh155p in vitro. [Relative (%)] Prp5p immunoprecipitation efficiencies (IP/input) were averaged from three independent repeats and normalized to the corresponding wild-type protein.

Figure 6.

Conformational changes of the prespliceosome are affected by interaction between Prp5p and Hsh155p. (A, left) Hsh155p mutants that enhanced in vitro Prp5p interaction had decreased in vivo affinity with Prp5p. (Right) In contrast, Hsh155p mutants that inhibited in vitro Prp5p interaction had no obvious altered in vivo affinity with Prp5p. (B) The DPLD motif mutant Prp5p that enhanced in vitro Hsh155p interaction had decreased in vivo affinity with Hsh155p. (C) Prp5p significantly associates with U1, U2 snRNAs, and pre-mRNA. (D) In the presence of the hsh155-H313S or hsh155-H331R allele, Prp5p's associations with U1, U2 snRNA, and pre-mRNA are significantly decreased but not in the presence of the hsh155-H331D or hsh155-K335N allele. (E) Disruption of complex conformation by high salt restores the interaction between Hsh155p and Prp5p. In vivo pull-down assays in A, B, and E were performed by HA beads against HA-Hsh155 and visualized by Western blotting. RT-qPCR assays in C and D were performed by Flag beads against Flag-Prp5p. [Relative (%)] Prp5 immunoprecipitation efficiencies (IP/Input) were averaged from three independent repeats and normalized to the corresponding wild-type protein. Brown and green labels represent two opposite groups of hsh155 alleles. (*) P < 0.05; (**) P < 0.01.

Figure 7.

Hsh155p/SF3B1 HEAT motif mutations alter its interaction with Prp5p and result in changes to splicing fidelity at the BS region. (A) Summary of phenotypes of selected Hsh155p/SF3B1 mutations from both cancers and yeast genetic screens. (B) Interaction between Prp5p and Hsh155p/SF3B1 promotes prespliceosome formation (complex A) and then leads to the release of Prp5p. In this model, mutations in Hsh155p or Prp5p that increase the Prp5p–Hsh155p interaction accelerate prespliceosome formation and, subsequently, fast release of Prp5p and also specifically suppress the splicing defects caused by suboptimal BS region substrates. Conversely, mutations that decrease the Prp5p–Hsh155p interaction slow down the formation of prespliceosomes and slow the release of Prp5p, thereby exacerbating the splicing defects of suboptimal BS substrates.