Background
Breast cancer is the one of the leading causes of death in females, second only to lung cancer. Gene expression and epigenetic profiling of breast cancer has shown that it is not a single disease, but rather a group of molecularly distinct neoplastic disorders [1]. There are five distinct molecular subtypes of breast cancer, which correlate with hormone responsiveness, patient prognosis, and response to therapy. Nonetheless, little is known about the initial, disease driving transcriptional and epigenetic changes, which promote the phenotypic outcomes in breast cancer.
Alternative splicing (AS) occurs in more than 90% of all human genes and increases the palette of proteins. AS of distinct isoforms is key in onset and progression of disease and a hallmark of cancer. Quaking STAR protein (QKI) is an RNA binding protein involved in RNA biogenesis such as alternative splicing, circular RNA production, protein translation and mRNA stability. QKI expression is found to be dysregulated in several cancers including colon, breast, prostate and lung cancer [2]. QKI has been shown to mediate alternative splicing of histone variant macroH2A, which regulate cancer cell proliferation [3].
Project: This master project will be a part of an ongoing project working on Quaking STAR protein in breast cancer. We aim to study the alternative splicing mediated by QKI variants in different subtypes of breast cancer. We will use CRISPR/Cas9 to generate an Auxin Inducible Degron (AID) system [4] to rapidly knock down QKI in breast cancer cell lines allowing for global RNA-seq analysis of splice variants affected. With the advantage to rapidly knock down QKI using AID system we will be able to investigate the role of QKI in global RNA biogenesis and avoid any secondary effects. The master student will work with a Marit Ledsaak (Senior engineer) in the Eskeland lab at IMB and will use various breast cancer cell lines to understand the role of this RNA binding protein QKI in epigenetic regulation of breast cancer malignancy.
Methods
The student will learn a wide range of biological/biochemical and molecular approaches used in our lab, like mammalian tissue culture, CRISPR/Cas9 technology, different knockdown methods including the Auxin inducible degron technology (AID) and RNAi, Cloning, Cell Transfection, Western blotting, RNA isolation, qRT-PCR, RNA-seq and bioinformatic data-analysis.
Graphical illustration
QKI is a splicing protein involved in alternative splicing of genes and also generates circular RNAs. The level of QKI increases circular RNA and promotes epithelial to mesenchymal transition (EMT). The masterstudent will use CRISPR to integrate AID-tag in breast cancer cell lines. Overview of AID system (Adapted from Natsume et al., 2016), in addition to the workflow of RNA-seq and Data-Analysis. When AID kd cells are generated, the student will generate RNA-seq and learn to do bioinformatic analysis of the datasets.
This master project is part of the Centre for Cancer Cell Reprogramming a Centre of Excellence at UiO (https://www.med.uio.no/cancell/english/). To learn more about us visit www.chromatome.no and see our movie: https://youtu.be/6yADYLbFEo4
References
- Fleischer T, Tekpli X, Mathelier A, Wang S, Nebdal D, Dhakal HP, et al. DNA methylation at enhancers identifies distinct breast cancer lineages. Nat Commun. Nature Publishing Group; 2017;8: 1379. doi:10.1038/s41467-017-00510-x
- Darbelli L, Richard S. Emerging functions of the Quaking RNA-binding proteins and link to human diseases. WIREs RNA. Wiley-Blackwell; 2016;7: 399–412. doi:10.1002/wrna.1344
- Novikov, L. et al. QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell proliferation. MOLECULAR AND CELLULAR BIOLOGY 31, 4244–4255 (2011).
- Natsume, T., Kiyomitsu, T., Saga, Y. & Kanemaki, M. T. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell reports 15, 210–218 (2016).