Poster abstracts
Poster number 54 submitted by James Hiznay
Further characterization of the roles of DDX41, a DEAD-box RNA helicase, in blood cancer and the spliceosome
James M. Hiznay (Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University), Chantana Polprasert (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Hideki Makishima (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Eckhard Jankowsky (Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Richard A. Padgett (Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University)
Abstract:
Members of the DEAD-box RNA helicase family have prominent roles throughout RNA metabolism. In addition to unwinding RNA duplexes, these proteins can remodel RNA-protein complexes and serve as scaffolds on which ribonucleoproteins assemble. Members of this protein family play a prominent role in the splicing of pre-mRNAs, where they serve as key components of the spliceosome. Sequencing of tumors from myelodysplastic syndrome (MDS) patients has revealed mutations—both germline and tumor-specific—in DDX41. MDS is a type of blood neoplasm that occurs in the elderly; it is typified by incomplete differentiation of erythrocytes, platelets, and certain leukocytes. A bevy of DDX41 mutations in MDS patients has been uncovered in the past three years. The most common DDX41 germline mutation causes a frame shift in the protein and results in a nonfunctional polypeptide. The most common tumor-specific mutation, R525H, affects the ATP-binding site and abrogates the ATPase activity of the protein. People with DDX41 mutations develop MDS younger and have a poorer prognosis than those without mutations. While DDX41 appears essential for cell growth, little is known of its role in the spliceosome or myeloid neoplasms. RNA-seq analysis of mutant patient tumors has shown a number of genes with altered spliced isoform ratios. Knockdown of DDX41 in cells results in the inhibition of splicing of a subset of introns. RNA CLIP-seq analysis of tagged DDX41 discovered preferential crosslinking to both U2 and U12 snRNAs as well as to 5’ and 3’ splice site regions of pre-mRNAs. Proteomic analysis of proteins co-precipitated using tagged DDX41 revealed numerous splicing factors, particularly from spliceosomal C complexes. Similar analyses using the R525H mutant protein showed reduced association with several key factors, including PRPF8 and SF3B1, which are also mutated in myeloid neoplasms. RNA co-immunoprecipitation experiments performed in an in vitro splicing assay with recombinant, tagged DDX41 evinced an enrichment of the lariat intron product. These data support our hypothesis that DDX41 is a core spliceosome component whose altered expression can affect the splicing of key tumor suppressor genes, which in turn may promote leukemogenesis.
Keywords: DDX41, RNA splicing, MDS