Principal Supervisor: Dr Olga Makarova, Department of Molecular and Cell Biology
Co-supervisor: Dr Andrey Revyakin and Dr Cyril Dominguez
PhD project title: Department of Molecular and Cell Biology
University of Registration: University of Leicester
Background. Sam68 is an RNA-binding protein that guides alternative splicing of transcripts involved in regulation of apoptosis and cell proliferation in response to extracellular signals. Also, Sam68 is a well-known proto-oncogene. It is ubiquitously expressed and has multiple interacting partners including transcription, splicing, RNA-export, and translation factors. However, despite of numerous studies, the mechanisms of Sam68 functions remain unclear. Therefore, elucidation of Sam68 interactome and targets genome-wide will enable further investigations into its potential as a drug-target in cancer therapies.
The goal of the project is to understand how Sam68 influences the formation of early splicing complexes and its role in transcription regulation.
Aims and Objectives.
- Identification of Sam68 interacting partners in mammalian cells by immunoprecipitation followed by mass spectrometry. For this, complexes will be purified via FLAG and biotin ligase (BirA) tags being integrated in the endogenous Sam68 gene. Also, the complexes will be isolated under different conditions including inhibition of transcription, splicing, translation and oxidative stress. These studies will be informative to the ongoing structural work on Sam68 led by Dominguez’s lab.
- Analysis of Sam68 functions by knockdown of the protein using cell line with Sam68-AID. Validation of the identified gene-targets will be carried out using RT-qPCR.
- Identification of the Sam68 binding sites genome-wide using CHIP and CLIP techniques.
- Determination of cellular localisation of Sam68 under different conditions using confocal and super-resolution single molecule microscopy. For this purpose, already generated Sam68-FLAG cell line will be used for treatments followed by visualisation of the protein relative to the transcription and splicing machineries using confocal microscopy. Importantly, Sam68 fluorescently tagged (HALO) will be used for single molecule co-localisation studies with Pol II and different splicing factors using the direct STORM super-resolution technique.
- Visualisation of Sam68 associated complexes using electron microscopy and identification of their structure.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- CRISPR/Cas9 gene editing
- Cell culture
- Isolation of protein complexes
- Mass spectrometry
- Western blotting
- Electron microscopy
- Structure determination