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Molecular Analyses of DDX41 in the Spliceosome and Myeloid Neoplasms

Hiznay, James Matthew

Abstract Details

2019, Doctor of Philosophy, Case Western Reserve University, Molecular Medicine.
Within the past decade, links between RNA splicing and myeloid neoplasms have emerged in the basic and clinical literature. DDX41 is a putative DEAD-box RNA helicase involved in the function of the spliceosome. Like several spliceosomal factors, mutations in DDX41 are observed in myelodysplastic syndrome (MDS). Uniquely, heterozygous frameshift DDX41 mutations can occur in the germ line of MDS patients with or without somatic missense mutations in the other allele. We cataloged MDS patients with somatic and germ line DDX41 mutations. Relative to MDS patients lacking a DDX41 mutation, our patient cohort had fewer co-mutations in epigenetic modulators, but co-mutations in DNA repair factors were increased. We examined the role of DDX41 perturbations on gene expression and splicing. We found that DDX41 binds to pre-mRNAs near splice sites and to some spliceosomal snRNAs, notably U2 and U12. Recombinant DDX41 protein was shown to have double-stranded RNA unwinding and ATP hydrolytic activities. Recombinant protein with the recurrent somatic mutation p.Arg525His (p.R525H) bound RNA about fourfold better but unwound RNA about fourfold slower than wild type. Nuclear extract created from DDX41-deficient leukemic cells spliced RNA approximately threefold slower than nuclear extract derived from HeLa cells. In cell lines, both knockdown of DDX41 and expression of p.R525H mutant DDX41 led to changes in splicing and expression in a variety of genes. These changes were similar to the changes seen in cell lines with mutations in the MDS-associated splicing factors SF3B1 and U2AF1. Global splicing analyses suggested that DDX41 delays intron removal, perhaps as part of a proofreading step, while the p.R525H mutant has a dominant negative stimulatory effect on splicing. We also saw that DDX41 perturbations affected the levels of many other spliceosomal factors, likely producing a cellular environment permissive for aberrant splicing events.
Richard Padgett, Ph.D. (Advisor)
Donal Luse, Ph.D. (Committee Chair)
Jaroslaw Maciejewski, M.D., Ph.D. (Committee Member)
Robert Silverman, Ph.D. (Committee Member)
George Stark, Ph.D. (Committee Member)
138 p.

Recommended Citations

Citations

  • Hiznay, J. M. (2019). Molecular Analyses of DDX41 in the Spliceosome and Myeloid Neoplasms [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1555347987446859

    APA Style (7th edition)

  • Hiznay, James. Molecular Analyses of DDX41 in the Spliceosome and Myeloid Neoplasms. 2019. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1555347987446859.

    MLA Style (8th edition)

  • Hiznay, James. "Molecular Analyses of DDX41 in the Spliceosome and Myeloid Neoplasms." Doctoral dissertation, Case Western Reserve University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1555347987446859

    Chicago Manual of Style (17th edition)