title: U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.
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.
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: T
authors.initials: T
authors.lastname: Okeyo-Owuor
authors.affiliation: 1] Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA [2] The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: White
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: R
authors.initials: R
authors.lastname: Chatrikhi
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Mohan
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kim
authors.affiliation: The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: M
authors.initials: M
authors.lastname: Griffith
authors.affiliation: The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: L
authors.initials: L
authors.lastname: Ding
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ketkar-Kulkarni
authors.affiliation: The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: J
authors.initials: J
authors.lastname: Hundal
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Laird
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Kielkopf
authors.affiliation: 1] Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA [2] The Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Ley
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Walter
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University, Saint Louis, MO, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Graubert
mesh_terms: D017398:Alternative Splicing; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D061251:Primary Cell Culture; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D014162:Transfection
chemical_list: D018952:Antigens, CD34; D009687:Nuclear Proteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human