Poster abstracts
Poster number 56 submitted by James Hiznay
The roles of DDX41 in the spliceosome and myeloid neoplasms
James M. Hiznay (Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH), Chantana Polprasert, Hideki Makishima (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH), Isabell Schulze, Carsten Muller-Tidow (Department of Hematology and Oncology, University of Halle, Halle, Germany), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH), Richard A. Padgett (Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH)
Abstract:
Myelodysplastic syndrome (MDS) encompasses a group of bone marrow failure disease characterized as hematological pre-malignancies that often further transform to acute myeloid leukemia (AML). A recent surprising finding in patients with MDS or AML is recurrent mutations of components of the spliceosome, the nuclear complex responsible for the process of pre-mRNA splicing during gene expression. Spliceosomal protein genes such as SF3B1, U2AF1, and SRSF2 are found to have somatic (i.e. tumor) mutations at specific “hot spots.” Whole exome sequencing of MDS patients’ genomes and bone marrow has identified novel mutations in another spliceosome factor gene named DDX41. In this case, both germline and somatic mutations were found. This unique combination results in lower expression of DDX41 and confers a more advanced stage of disease and a lower patient survival outcome. DDX41 is a member of the DEAD-box family of RNA helicases, many of which are involved in splicing. Although proteomics has identified DDX41 as a component of the spliceosome catalytic core (complex C), no information is available on the exact role the protein plays in splicing or on its interaction partners in the spliceosome. We hypothesize that DDX41 interacts with one or more of the complex C snRNAs (U2, U5, U6) and/or with the splice sites. Our work has focused on determining the RNA interaction sequences of wild type and mutant DDX41 in vivo using RNA-protein crosslinking, complex immunoprecipitation, and high-throughput sequencing (collectively known as CLIP-seq). For this study, we have generated 293 cells expressing V5-tagged DDX41. Observed sensitivity to RNase A titration following crosslinking adds credence to the hypothesis that DDX41 is an RNA-binding protein. Preparation of cDNA libraries generated from RNA isolated during a CLIP is currently underway. Future work on this project includes the analysis of changes in RNA binding partners of mutant DDX41 as well as the generation of depleted nuclear extracts for in vitro biochemical studies.
References:
Maciejewski JP, Padgett RA. Br J Haematol. 2012 Jul;158(2):165-73.
Makishima H et al. Blood. 2012 Apr 5;119(14):3203-10.
Padgett RA. Trends Genet. 2012 Apr;28(4):147-54.
Keywords: DDX41, Pre-mRNA splicing, Myeloid neoplasm