Intrinsically disordered regions (IDRs) of protein play crucial roles in almost all cellular functions. Still, the molecular mechanisms that govern their functions remains largely unknown.
Sam68 is an oncogenic RNA binding protein that regulates multiple aspects of gene expression and is strongly controlled by signalling pathways through post-translational modifications (PTMs). Sam68 contains a classical folded RNA binding domain flanked by N-terminal and a C-terminal intrinsically disordered regions (IDRs). While these IDRs are crucial for the function of Sam68 and are targets of multiple post-translational modifications, the molecular mechanisms of their contribution remains unknown.
Our recently published data (Malki et al, NAR, 2022) demonstrated that the Nter and Cter IDRs of Sam68 have the ability to bind RNA specifically and that phosphorylation of a single threonine inhibits their RNA binding ability and consequently the cellular functions on the protein. In parallel, our partner, Yannick Benoit, has identified a small molecule that bind specifically to Sam68 IDRs, leading to decreased tumour incidence and invasiveness in mice and in patient-derived colorectal cancer stem cells organoids.
Specifically, we aim to answer the following three questions:
1- What are the molecular bases of the specific RNA recognition by the N- and C-terminal IDRs of Sam68?
2- How does phosphorylation at a single amino acid or binding of a peptidomimetic compound affect the RNA binding ability of these IDRs?
3- How do the STAR domain and the IDRs of Sam68 cooperate to bind RNA targets with high affinity?
We will answer these questions by combining the team expertise in structural and biophysical methods (NMR, FCS, FRET, SAXS) with molecular dynamics to decipher the structural properties of these regions free, in complex with RNA and following phosphorylation.
Techniques that will be undertaken during the project
• Protein expression (E. coli) and purification
• Nuclear Magnetic Resonance (NMR)
• Fluorescence Correlation Spectroscopy (FCS)
• Single-molecule Foerster Resonance Energy Transfer (sm-FRET)
• Small-Angle X-ray scattering (SAXS)
• Molecular dynamics simulations
Enquiries
Project Enquiries to cd180@le.ac.uk
Programme enquiries to mibtp-admin@leicester.ac.uk
To apply please refer to
https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp
