DDX41 Helicase in P-body Formation and Myeloid Malignancies
Date
2025-01-03
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Masters
Abstract
DDX41 is a member of the DEAD-box helicase family. Mutations in DDX41 cause myeloid malignancies, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MDS is characterized by unsuccessful hematopoiesis and peripheral blood cytopenia, one-third of MDS patients will progress to AML. AML is cancer in the bone marrow and the spread of immature myeloid cells. DDX41 has been implicated in innate immunity and mRNA splicing. The predominant mutation in DDX41 is R525H; however, its molecular pathogenesis in MDS/AML remains unknown. Processing bodies (PBs) are cytoplasmic ribonucleoprotein granules primarily comprised of decapped mRNAs complexed with various proteins associated with translation repression and mRNA decay. Although, PBs exist in normal cellular conditions, they increase in formation under stress conditions. PB dysregulation has been associated with various diseases and cancers, including MDS/AML. Proteins EDC4 (Enhancer of mRNA decapping 4), 4E-T (4E-transporter), LSM14A (LSM14A mRNA processing body assembly factor), and DCP1A (mRNA-decaping enzyme 1A) are PB components. Using DDX41-knockout (KO) cell lines and PB markers EDC4 and DCP1A, I found that DDX41-depletion reduces PB formation under stress conditions, namely sodium arsenite–induced oxidative stress and glucose starvation. However, DDX41 predominantly does not localize in the PBs, implying that DDX41 indirectly affects PBs. Western blot analysis revealed that DDX41-deficiency does not significantly impact the PB protein level. Examining the expression of endogenous DCP1A and exogenous GFP-tagged 4E-T found that the deficiency of DDX41 affected the endogenous foci formation but not GFP-tagged 4E-T, which indicates its potential role in mRNA splicing. Using qRT-PCR, I determined that DDX41 depletion can influence the mRNA levels of EDC4, 4E-T, and DCP1A; however, these effects were specific to the gene, primer location, and the type of stress applied. Furthermore, semi-quantitative PCR results revealed that the splicing variants of PB transcripts without stress treatment were altered in DDX41-deficient cells, reinforcing that DDX41 is involved in mRNA splicing, though this could be primer location specific. Lastly, we reconstituted DDX41-KO cells with DDX41-WT and DDX41-R525H genes and observed that the R525H mutant lead to an increase in PB formation. Taken together, our results demonstrate that DDX41 plays a role in PB homeostasis by acting as a PB accessory component, and its dysregulation may lead to the development of myeloid malignancies.
Description
Keywords
DDX41, P-bodies, MDS, AML, DEAD-box, helicase, R525H, cellular stress, myeloid malignancies, sodium arsenite, glucose starvation
Citation
Degree
Master of Science (M.Sc.)
Department
Biochemistry, Microbiology and Immunology
Program
Biochemistry