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Oncogenes, Fusion Genes and Tumor Suppressor Genes

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing

Leukemia volume 29, pages 909–917 (2015)Cite this article

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Abstract

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

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Acknowledgements

This work was supported by Barnes-Jewish Hospital Foundation (7603-55 to BSW), an NIH/NCI SPORE in Leukemia (P50CA171063 to TAG, MJW and BSW), an NIH/NCI grant (1K12CA167540 to BSW), a Clinical and Translational Award from the NIH National Center for Advancing Translational Sciences (UL1 TR000448 to BSW), a Howard Hughes Medical Institute Physician-Scientist Early Career Award and Leukemia and Lymphoma Society Scholar Award (to MJW), a Leukemia and Lymphoma Society Translational Research Award (to TAG), and NIH R01 GM070503 (to CLK). Technical assistance was provided by the Alvin J. Siteman Cancer Center High Speed Cell Sorting core (supported in part by P30CA91842), Molecular Microbiology Imaging Facility (MMIF), the Genome Technology Access Center (GTAC) and the Genome Institute. Additional technical support was provided by Masayo Izumi. We are grateful to Dr Christopher R Cabanski for assistance with edgeR and Dr Cara Lunn Shirai for helpful scientific discussions.

Author Contributions

The study was designed by TOO, TAG and MJW; RNA affinity assays were performed by KML, RC and CLK; sub-cellular localization and junction validation experiments performed by TOO; minigene assays were performed by DM, TOO and SK; Bioinformatics analysis was performed by BSW, MG, LD, SKK and JH; the manuscript was written and edited by TOO, BSW, MG, TJL, CLK, TAG and MJW; all co-authors reviewed and approved the submission.

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  1. T A Graubert

    Present address: 4Current address: Massachusetts General Hospital Cancer Center, Boston, MA, USA.,

Authors and Affiliations

  1. Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA

    T Okeyo-Owuor, B S White, D R Mohan, S Kim, S Ketkar-Kulkarni, T J Ley, M J Walter & T A Graubert

  2. The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA

    B S White, M Griffith, L Ding, J Hundal & T J Ley

  3. Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA

    R Chatrikhi, K M Laird & C L Kielkopf

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  1. T Okeyo-Owuor
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Correspondence to T A Graubert.

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Okeyo-Owuor, T., White, B., Chatrikhi, R. et al. U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing. Leukemia 29, 909–917 (2015). https://doi.org/10.1038/leu.2014.303

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