Optimizing the Conditions to Identify the DDX41 Interactome by BioID
| dc.contributor.advisor | Wu, Yuliang | |
| dc.contributor.advisor | Lukong, Kiven Erique | |
| dc.contributor.committeeMember | Wang, Hong | |
| dc.contributor.committeeMember | Bull, Harold | |
| dc.creator | Charaya, Ananaya | |
| dc.creator.orcid | 0009-0001-2352-6131 | |
| dc.date.accessioned | 2024-11-15T20:23:21Z | |
| dc.date.available | 2024-11-15T20:23:21Z | |
| dc.date.copyright | 2024 | |
| dc.date.created | 2025-06 | |
| dc.date.issued | 2024-11-15 | |
| dc.date.submitted | June 2025 | |
| dc.date.updated | 2024-11-15T20:23:21Z | |
| dc.description.abstract | DDX41 (DEAD-box 41) protein, an RNA helicase, is involved in several processes, including genome stability, mRNA splicing, ribosome biogenesis, translation, cell differentiation, and innate immunity. Mutations in DDX41 cause myeloid malignancies, especially myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our preliminary data showed that DDX41 experiences dynamic changes, including protein level and sub-cellular localization, when the cells are exposed to distinct stress conditions, including microbial infection (Herpes simplex virus) and radiation exposure (infrared radiations). However, the molecular mechanisms regulating DDX41 expression and sub-cellular localization are unknown. We hypothesize that DDX41-interacting proteins may regulate the dynamics of DDX41. To identify the interacting proteins of DDX41 in living cells, we employed a proximity-dependent biotin identification (BioID) approach; hence, we used an improved biotin ligase, TurboID. We constructed a DDX41-TurboID fusion construct, where DDX41 is tagged with 3xFlag at its N-terminus and TurboID ligase at its C-terminus and a control construct that contains 3xFlag and TurboID. By transfection in HEK293T and HeLa DDX41 knockout cells, I observed the protein expression and nuclear localization for DDX41-TurboID fusion proteins and cytoplasmic localization of the 3xFlag-TurboID (control) proteins by Western blot and immunofluorescence microscopy respectively. I found that the labelling time of TurboID ligase of 30 min and a biotin concentration of 50 µM were optimal for the biotinylation activity of the TurboID ligase on proxisomes and hence the biotinylation of potential DDX41-interacting proteins. I optimized the immunoprecipitation (IP) of biotin-treated BioID fusion proteins and DDX41-interactome using Flag and streptavidin conjugated to protein A/G agarose beads as well as the streptavidin sepharose beads. Western blots validated the optimization of the IP, resulting in the presence of a potential biotinylated DDX41-interacting protein. In inference, we have constructed and validated the expression and localization of BioID fusion constructs and optimized the conditions to identify the interactome of DDX41. In the future, mass spectrometric analysis will facilitate the identification of the interacting proteins of DDX41, and the validation of the DDX41-interacting proteins will lead us to a better understanding of the regulatory pathways and potential druggable targets for the treatment of DDX41-mutated disorders. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/16254 | |
| dc.language.iso | en | |
| dc.subject | DDX41 | |
| dc.subject | BioID | |
| dc.subject | Leukemia | |
| dc.title | Optimizing the Conditions to Identify the DDX41 Interactome by BioID | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Biochemistry, Microbiology and Immunology | |
| thesis.degree.discipline | Biochemistry | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |