Myelodysplastic Syndrome-Associated SRSF2 Mutations Cause Splicing Changes by Altering Binding Motif Sequences

So Masaki, Shun Ikeda, Asuka Hata, Yusuke Shiozawa, Ayana Kon, Seishi Ogawa, Kenji Suzuki, Fumihiko Hakuno, Shin-Ichiro Takahashi, Naoyuki Kataoka, So Masaki, Shun Ikeda, Asuka Hata, Yusuke Shiozawa, Ayana Kon, Seishi Ogawa, Kenji Suzuki, Fumihiko Hakuno, Shin-Ichiro Takahashi, Naoyuki Kataoka

Abstract

Serine/arginine-rich splicing factor 2 (SRSF2) is a member of the SR protein family that is involved in both constitutive and alternative mRNA splicing. Mutations in SRSF2 gene are frequently reported in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). It is imperative to understand how these mutations affect SRSF2-mediated splicing and cause MDS. In this study, we characterized MDS-associated SRSF2 mutants (P95H, P95L, and P95R). We found that those mutants and wild-type SRSF2 proteins showed nuclear localization in HeLa cells. In vitro splicing reaction also revealed that mutant proteins associated with both precursor and spliced mRNAs, suggesting that the mutants directly participate in splicing. We established the human myeloid leukemia K562 cell lines that stably expressed myc-tagged wild-type or mutant SRSF2 proteins, and then performed RNA-sequence to analyze the splicing pattern of each cell line. The results revealed that both wild-type and mutants affected splicing of approximately 3,000 genes. Although splice site sequences adjacent to the affected exons showed no significant difference compared to the total exons, exonic motif analyses with both inclusion- and exclusion-enhanced exons demonstrated that wild-type and mutants have different binding sequences in exons. These results indicate that mutations of SRSF2 in MDS change binding properties of SRSF2 to exonic motifs and this causes aberrant splicing.

Keywords: EZH2 (enhancer of zeste homolog 2); SRSF2; aberrant splicing; exonic splicing enhancer; myelodysplastic syndrome; splicing.

Figures

FIGURE 1
FIGURE 1
MDS-causing SRSF2 mutant proteins are localized in the nuclei and associate with pre-mRNA and mRNA in vitro. (A) HeLa cells were transfected with expression vectors encoding either wild-type or mutant SRSF2 proteins (P95H, P95L, and P95R) fused with myc-tag. (B)In vitro splicing reaction in the presence of either Flag-tagged wild-type SRSF2 protein or mutant proteins followed by immunoprecipitation of RNAs by anti-Flag M2 antibody. (C) Immunoprecipitation of SRSF2 proteins by anti-Flag antibody from in vitro splicing reaction. Five percent of total reaction mixture was used as an input. The anti-Flag M2 antibody was used for detection of Flag-tagged SRSF2 proteins.
FIGURE 2
FIGURE 2
Analyses of splicing changes in K562 cells stably expressing either wild-type or mutant SRSF2 protein. (A) Immunoblotting analyses of K562 cell lines stably expressing myc-tagged wild-type or mutant SRSF2 proteins. A myc-vector was used as a negative control. (B) Numbers of reads from RNA sequence analyses and mapping percentage of sequence tags. (C) Numbers of altered splicing events and genes identified by comparing with myc-vector transfected cells. (D) Venn diagram comparing the differentially spliced genes among myc-vector, WT and three SRSF2 mutants expressing cells.
FIGURE 3
FIGURE 3
SRSF2 mutants promote exon 9.5 of EZH2 gene through binding to CCWG motif. (A) Sequence logos for the motifs enriched in inclusion-type exons with WT, P95H, P95L, and P95R are presented. (B) Sequence logos for the motifs enriched in inclusion-type exons with WT, P95H, P95L, and P95R are shown. (C) Sequence of exon 9.5 (pseudo exon) in human EZH2 gene. In frame stop codons are underlined. CCWG motif, found with SRSF2 P95H and P95R proteins in (B), are indicated with open boxes. (D) Splicing pattern of EZH2 pre-mRNA in myc-vector, SRSF2 WT, P95H, P95L, and P95R are indicated. The ratio of exon9.5 included product in total splicing products is shown beneath the panel. The schematic representations of PCR products are shown at the right side of the panel. RT, Reverse Transcriptase. M, DNA size marker.
FIGURE 4
FIGURE 4
A model for splicing changes caused by SRSF2 mutations in the patients. (Left) SRSF2 wild-type protein binds to purine-rich exonic splicing enhancer for promotion of exon recognition. (Right) SRSF2 mutants, SRSF2 P95H and P95R, preferentially bind to CCWG motif-containing exons to promote aberrant splicing.

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