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How do RNA-Binding Proteins control splice site selection?

Primary Supervisor: Dr Cyril Dominguez, Department of Molecular and Cell Biology

Secondary supervisor: Professor Ian Eperon

PhD project title: How do RNA-Binding Proteins control splice site selection?

University of Registration: University of Leicester

Project outline:

This PhD project is part of a large multi-disciplinary programme, funded by a prestigious BBSRC sLoLa grant, that involves five research laboratories with expertise spanning physical and organic chemistry, engineering, structural biology, biophysics and biochemistry. The overall aim is to understand the complex mechanisms by which swarms of RNA-binding proteins influence and regulate alternative splicing.

More than 90% of human genes can and do express multiple proteins. This is achieved by a process called alternative RNA splicing, which is an essential step in gene expression in mammals. In human, more than 100,000 proteins are produced from only 20,000 genes. For example, neurexin 3 alone is believed to express 1,728 different proteins with different synaptic functions from one pre-mRNA sequence.

Alternative splicing dictates which protein to express; this varies between tissues, development stages or in response to extracellular environment and the choices made affect processes ranging from memory and differentiation to death and disease.

Alternative splicing is regulated by several hundred RNA-binding proteins in the nucleus. Through binding to the pre-mRNA, these proteins will compete or cooperate to induce the inclusion or exclusion of certain exons. However, the molecular mechanisms governing these regulatory events are still largely unknown.

Current methods have led to an understanding of the RNA-binding behaviour of domains of these proteins, but they have led to an impasse in which a pre-mRNA appears to be affected by more proteins than can physically be bound at any one time and which have conflicting effects upon the usage of sites or exons. In order to better understand splicing regulation, we need to understand the timing and permitted combinations of proteins bound and their dynamics, together with their interactions when bound and their effects on the biophysical properties of the RNA-protein complex. This is one of the great frontiers in gene expression.

The specific aims of this PhD project are to characterise the cooperativity or competition among proteins binding to the RNA and its effect on protein and RNA structures and dynamics. For this, fragments of the pre-mRNA will be 15N/13C-labelled and heteronuclear NMR spectra of the RNA will be measured with and without unlabelled proteins (full-length or isolated RNA binding domains (RBDs), expressed and purified from either E. coli or HEK293 cells). This will reveal if the RNA adopts secondary structures, provide a fingerprint for each protein-RNA interaction and indicate whether the protein affects the RNA structure. We will then add pairs of proteins to the RNA and investigate whether both proteins bind the same RNA or compete directly for binding. Similar experiments will be done with labelled proteins to investigate whether they interact with each other upon RNA binding. Importantly, all these experiments will be done in nuclear extracts to mimic the cellular environment of the proteins. The most interesting cooperative complexes will be further studied structurally by X-ray crystallography, NMR and/or SAXS and biophysically by ITC and fluorescence anisotropy. The dynamics of protein interactions will also be investigated by NMR.

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing & Diet and Health. Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

  • RNA and protein expression and purification
  • High-resolution NMR in nuclear extracts
  • X-ray crystallography
  • Isothermal Titration calorimetry
  • Fluorescence spectroscopy

Contact: Dr Cyril Dominguez, University of Leicester