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Chen, Richard

University of British Columbia

APEX2 proximity labeling was originally developed to capture the proteomes of different subcellular compartments. While its ability to study specific protein-protein interactions was initially unclear, this application is particularly compelling. This thesis presents an optimized sample preparation protocol for APEX2 proximity labeling, tailored to identify interactors of specific proteins of interest. The quantitative accuracy of the proximity labeling workflow was improved through changes in protein extraction and purification for mass spectrometry, as well as the analysis of mass spectrometry data. This optimized protocol will be useful for future proximity labeling studies of target proteins. CDK12 and CDK13 are kinases implicated in transcription, RNA processing, the expression of DNA damage response genes, and genome stability in various cancers (e.g., ovarian, breast, prostate, and endometrial). Although the protein-level mechanisms of CDK12 and CDK13 are not fully understood, pharmaceutical inhibitors targeting these kinases are currently in development for cancer chemotherapy. To better understand the molecular functions of these kinases, proximity labeling data was generated using the optimized protocol for CDK12, CDK13, and their activating cyclin, CCNK. The data suggests that CDK12 and CDK13 may be recruited for transcription and RNA processing through phase separation with other RNA processing proteins. WAC and TTC14 were identified as novel interacting partners. Also proposed are possible mechanisms of genomic tandem duplication formation in BRCA1-deficient and CDK12-deficient cells.

Text

2024-12-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89893

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/