Poster abstracts
Poster number 64 submitted by Noah Daniels
Investigating the Role of DDX41 in pre-messenger RNA Splicing and Leukemogenesis
Noah J. Daniels (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), James M. Hiznay (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), Courtney E. Hershberger, William M. DiPasquale, Devlin C. Moyer (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), Sukanya Srinivasan, Eckhard Jankowsky (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH), Richard A. Padgett (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH)
Abstract:
DEAD-box RNA helicases are a highly conserved family of proteins involved in nearly all aspects of RNA metabolism, including several members with crucial roles in pre-messenger RNA splicing. Whole exome sequencing has identified DDX41, a member of this family, as recurrently mutated in myelodysplastic syndrome (MDS) patients. MDS is a bone marrow neoplasm that results from a buildup of precursor cells giving rise to subsequent defects in the myeloid lineages. Germline frameshift DDX41 mutations potentially causing DDX41 hemizygosity throughout life have been identified in numerous families with histories of MDS and leukemia. Nearly half of these individuals will acquire a recurrent, somatic, missense mutation, resulting in the conversion of arginine 525 to histidine (R525H) in the highly conserved helicase domain of the second DDX41 allele. Biochemical assays in vitro with recombinant DDX41R525H displayed slightly tighter binding of double stranded RNA and a 3-fold decrease in RNA duplex unwinding compared with DDX41WT protein; this suggests a hypomorphic function. Proteomic analyses have classified DDX41 as a member of the catalytic core of the spliceosome but its role in splicing is still undefined. Analysis of RNA cross-linking-immunoprecipitation-high-throughput-sequencing data demonstrated that DDX41 binds preferentially to exons and splice sites of pre-mRNAs. Furthermore, DDX41 binds to both major and minor class spliceosomal snRNAs. RNA-seq analyses of DDX41WT and DDX41R525H over-expression or DDX41 knockdown human cells have revealed subtle-yet global-effects on pre-mRNA splicing. For future studies, we are generating an auxin-inducible degron system to promote complete degradation of DDX41 protein to determine the function of DDX41 in splicing using in vitro splicing assays. These data support our hypothesis that DDX41 is a component of the spliceosome and plays an important role in pre-mRNA splicing. Mutations and/or deletions of DDX41 may result in the aberrant splicing of key leukemic drivers; in turn, this results in leukemogenesis.
Keywords: myelodysplastic syndrome, DEAD-box RNA helicases, pre-mRNA s