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. 2015 Aug 25;112(34):E4726-34.
doi: 10.1073/pnas.1514105112. Epub 2015 Aug 10.

Disease-associated Mutation in SRSF2 Misregulates Splicing by Altering RNA-binding Affinities

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Free PMC article

Disease-associated Mutation in SRSF2 Misregulates Splicing by Altering RNA-binding Affinities

Jian Zhang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

Keywords: leukemia; myelodysplastic syndromes; pre-mRNA splicing; serine/arginine-rich proteins; spliceosome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Validation of splicing targets of mutSRSF2. (A, Lower Left) P95H allele expression in the mutant CRISPR clones. (Lower Right) Expression of SRSF2 in 293T cells either mock transfected (–) or transfected with plasmid encoding HA-tagged WT (W) or P95H mutSRSF2 (P). In the immunoblot analysis, the mAb104 antibody was used to detect endogenous and HA-tagged SRSF2. (Upper) RT-PCR products of exon inclusion and exclusion isoforms of ATF2. (BG) RT-PCR products of splicing isoforms of MELK (B), PFKM (C), CDK5RAP2 (D), ARMC10 (E), DGUOK (F), and WDR45 (G). In all panels, each CRISPR clone was considered as one independent experiment. Because we had four WT and four mutSRSF2 CRISPR clones, n was 4 (as indicated). For 293T cells, three independent transfection experiments were performed (n = 3). Rounded percentages of exon inclusion or exclusion and SDs are shown as indicated. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. S1.
Fig. S1.
Confirmation of SRSF2 mutations in CRISPR clones by DNA sequencing. P95H and synonymous mutations were introduced to SRSF2 in K562 cells by electroporation of the SRSF2 gRNA CRISPR/Cas9 vector along with either the P95H mutant or WT SRSF2 ssODN. Synonymous mutations were used to create restriction enzyme sites [NaeI (C→G) in WT cells and HaeII (C→G) in mutant cells], to facilitate identification of knock-in clones by restriction digest, or to disrupt the protospacer-adjacent motif (PAM) site (G→A) to prevent gRNA/Cas9 from recognizing and cutting the newly introduced mutant allele. Genomic DNAs were extracted from positive clones, and the SRSF2 mutations were confirmed using PCR and DNA sequencing. The P95H mutation (C→A) and synonymous mutations are indicated above the sequencing chromatograms. (A) K562 parental cells. (B) CRISPR clones with WT SRSF2. (C) CRISPR clones with P95H mutSRSF2.
Fig. S2.
Fig. S2.
Total mRNA levels of SR proteins and hnRNP splicing factors were comparable in WT and mutSRSF2 CRISPR clones. (Upper) Total mRNA levels of SRSF2 and 11 other SR proteins in K562 parental cells and WT and mutSRSF2 CRISPR cells. (Lower) Total mRNA levels of 16 major hnRNP splicing factors. Except that there were small but significant differences for HNRNPA2B1 (∼7% decrease in mutant vs. WT SRSF2 cells, P = 0.043) and HNRNPC (∼19% decrease, P = 0.008), the mRNA levels of the other 14 hnRNP splicing factors and all 12 SR proteins did not show significant changes between WT and mutant SRSF2 cells. Note that expression of SRSF8 and SRSF12 was barely detectable.
Fig. S3.
Fig. S3.
Validation of additional splicing targets of mutSRSF2. Experiments were performed as in Fig. 1. The panels show the RT-PCR products of splicing isoforms of CRAT (A), SLC25A26 (B), ABI1 (C), FYN (D), CDC45 (E), and EZH2 (F, see ref. 7). *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. S4.
Fig. S4.
Sequence logo of the conserved motif in SRSF2-binding sites. A list of 109 published SRSF2-binding sites was compiled (see text). A 10-nt motif was identified in 88 of the 109 sites using MEME. A sequence logo of the motif was generated using WebLogo (weblogo.berkeley.edu/logo.cgi).
Fig. 2.
Fig. 2.
Distinct sequence motifs are enriched in the regulated cassette exons. The relative frequencies of occurrence of highly enriched 4-mers (A) and 5-mers (B) in the more-included or more-excluded exons (mutant vs. WT) are shown.
Fig. S5.
Fig. S5.
Enriched sequence motifs do not show obvious positional effects. Locations of UCCWG and UGGWG sequences relative to exon boundaries do not show an obvious uneven distribution. Data are shown as percentile of exon length.
Fig. 3.
Fig. 3.
The P95H mutation alters binding of SRSF2 to distinct RNA sites. The indicated SRSF2-binding sites (10-nt RNAs) from target transcripts were incubated with increasing concentrations of His6-tagged WT or mutant (P95H) SRSF2 (amino acids 1–101), and protein–RNA complexes were resolved from free oligonucleotides by gel electrophoresis. Experiments were performed with two independent protein preparations. Final concentrations of recombinant SRSF2 proteins in the gel shift assays were (A) 0, 0.18, 0.24, 0.30, 0.36, 0.42, and 0.48 μM; (B) 0, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 μM; (C) 0, 0.06, 0.12, 0.18, 0.24, 0.30, and 0.36 μM; (D) 0, 0.12, 0.18, 0.24, 0.30, 0.36, and 0.42 μM; (E) 0, 0.36, 0.48, 0.60, 0.72, 0.84, and 0.96 μM; (F) 0, 0.12, 0.18, 0.24, 0.30, 0.36, and 0.42 μM; and (G) 0, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 μM. The apparent Kds are shown in each panel.
Fig. S6.
Fig. S6.
Purified His6-tagged WT and mutSRSF2 (amino acids 1–101). Two micrograms of His6-tagged WT (W) or P95H mutant (P) SRSF2 (amino acids 1–101) were subjected to 15% SDS/PAGE and Coomassie staining.
Fig. 4.
Fig. 4.
Occurrences of putative SRSF2 sites in the cassette exons. Numbers of cassette exons (either more included or more excluded in mutant vs. WT SRSF2 cells) harboring UCCWG, UGGWG, and/or other putative sites are shown. Note that 66 exons do not harbor UCCWG, UGGWG, UGCWG, or UCGWG sites. These may be false positives of rMATS (66/374 = 18%), as is consistent with the ∼80% validation rate (∼20% false rate).
Fig. 5.
Fig. 5.
Exon inclusion/exclusion in minigene reporter assays. Splicing reporter minigenes were cotransfected with plasmid expressing WT (W) or P95H mutSRSF2 (P) into 293T cells. Exon inclusion and exclusion isoforms were examined by RT-PCR. Three independent experiments were performed (n = 3). (A) MELK cassette exon minigene containing the native 10-nt site and its mutant minigenes containing mutated 10-nt sites as indicated. The 5-nt motif sequences were capitalized. Note that a cryptic 3′ splice site was detected in the UUUUU1 minigene (last two lanes). (B) The ATF2 cassette exon minigene containing the native 10-nt site and its mutant minigenes containing mutated 10-nt sites as indicated. The 5-nt motif sequences were capitalized. In the last two lanes, the PCR products of the exon inclusion isoform (indicated by an arrow) appeared to migrate slightly more slowly than the PCR products in other lanes. However, DNA sequencing indicated that the PCR products in the last two lanes had exactly the same 5′ and 3′ splice sites as the PCR products in the first two lanes.
Fig. 6.
Fig. 6.
Interactions of SRSF2 with U2AF1, snRNP70, and SF3B1 are unaffected by the P95H mutation. Coimmunoprecipitation was performed, in the presence of RNase, with anti-HA rabbit polyclonal antibody using extracts of 293T cells either mock-transfected (–) or transfected with plasmid expressing HA-tagged eGFP (G), WT (W), or P95H mutSRSF2 (P). The heavy and light chains of anti-HA polyclonal antibody were detected in the anti-ACTIN (rabbit) and anti-U2AF1 (rabbit) immunoblots due to the use of the anti-rabbit secondary antibody. The mAb104 antibody was used to detect endogenous and HA-tagged SRSF2 proteins.
Fig. S7.
Fig. S7.
A hypothetical model of the functional mechanism of mutSRSF2. Pro-95 in WT SRSF2 (shown as a light green oval) forms a stacking interaction (dashed vertical line) with both the second cytosine in UCCWG sites (A) and the second guanine in UGGWG sites (B). His-95 in mutSRSF2 (shown as a light red oval) may form an H-bond (solid vertical line) with the second cytosine in UCCWG sites (C). An H-bond is generally stronger than a stacking interaction. Because the second guanine in the UGGWG sites is in syn-conformation, it is not possible for His-95 in mutSRSF2 to form an H-bond, yielding weaker binding to UGGWG sites (shown as an X) (D).

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