The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes
Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood
Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood
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Research Article Hematology

The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes

Abstract

Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Authors

Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood

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Figure 3

U2AF1S34F differentially alters splicing of target genes in erythroid and granulomonocytic colonies.

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U2AF1S34F differentially alters splicing of target genes in erythroid a...
(A) Quantification of U2AF1 WT (TCT) and S34F mutant (TTT) mRNA in erythroid and granulomonocytic colonies, determined by pyrosequencing. (B and C) Aberrant splicing events associated with U2AF1S34F, including breakdown by event type, in erythroid and granulomonocytic colonies for (B) U2AF1S34F versus EV and (C) U2AF1S34F versus U2AF1WT. (D) Sequence logos for 3′ splice sites of cassette exons that were unaffected (top row), more included (middle row), or more skipped (bottom row) in response to U2AF1S34F compared with U2AF1WT. (E) Distribution of exon inclusion and skipping events within the total number of regulated cassette exon events in the comparison of U2AF1S34F versus U2AF1WT in erythroid and granulomonocytic colonies. (F) Venn diagram showing the overlap among the genes that contained aberrant splicing events induced by U2AF1S34F in erythroid and granulomonocytic colonies in our study. (G–J) Venn diagrams showing the overlap among the genes that contained aberrant splicing events induced by U2AF1S34F in different RNA-seq data sets: (G) transgenic mouse CMPs expressing U2AF1S34F and erythroid colonies and granulomonocytic colonies in our study; (H) TCGA AML patient samples with U2AF1 S34 mutations and erythroid colonies and granulomonocytic colonies in our study; (I) U2AF1S34F MDS CD34+ bone marrow cells (versus MDS cases without splicing factor gene mutations) and erythroid colonies and granulomonocytic colonies in our study; and (J) U2AF1S34F MDS CD34+ bone marrow cells (versus healthy controls) and erythroid colonies and granulomonocytic colonies in our study. Results in panel A are shown as the mean ± SEM and were obtained from 3 independent experiments. P values in panel B and C were calculated by Fisher’s exact test with Bonferroni’s correction. *P < 0.05 and **P < 0.01. A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; MXE, mutually exclusive exon; RI, retained intron; SE, skipped (cassette) exon.