Structure and dynamics of the intrinsically disordered regions of the RNA binding protein Sam68: implication for RNA binding and phosphorylation.

About the Project

A large proportion of the human proteome is composed of unstructured regions, termed intrinsically disordered regions (IDRs). Over recent years, it has been demonstrated that these regions are crucial for almost all cellular functions but structural studies of these IDRs to unravel the molecular mechanisms of their function remain largely unknown.

A typical example is the oncogenic RNA-binding protein Sam68, a multifunctional protein contributing to regulation of RNA metabolism, and signal transduction that is often overexpressed in many types of cancers. While the role of Sam68 central RNA binding domain in RNA recognition is well characterized, it is currently unknown how Sam68 N-terminal (Nter) and C-terminal (Cter) IDRs contributes to RNA binding and function of the protein, although these regions are targeted by multiple post-translational modifications (PTMs) that modulate Sam68 functions. By investigating the role of PTMs in regulating the cellular functions of Sam68, we have demonstrated that Sam68 Nter and Cter IDRs bind RNA specifically and that phosphorylation of these regions by Cdk1 modulates their RNA binding and the cellular functions of the protein. How do these regions bind specifically RNA and how phosphorylation of a single amino acid affect the interaction are still unknown. Our hypothesis is that Sam68’s IDRs adopt transient structural features that are crucial for specific RNA recognition and that phosphorylation of these regions modulate these features, preventing RNA binding. Our hypothesis is supported by strong preliminary NMR data.

We will combine mutiple biochemical biphysical and structural techniques to decipher the structural properties of Sam68 IDRs either free, in complex with RNA or following phosphorylation by CDK1.

This multi-disciplinary project will provide unique structural and mechanistic insights into RNA binding properties of IDPs/IDRs.

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

To apply please use the application link at the bottom of this web page

Funding Notes

This project is only available on a self funded basis or if you have your own sponsorship.