How do RNA-binding proteins control splice site selection? A multi-disciplinary approach.

Almost every protein-coding gene in vertebrates can and does express multiple proteins. This is achieved primarily by RNA splicing, which is an essential step in gene expression in mammals and generates incredible diversity. There is a good correlation between the levels of alternative splicing and an organism’s complexity. Indeed, it is widely held that the development of alternative splicing has been a key enabler of the evolution of complex organisms. The numbers of isoforms peak in neuronal tissue; neurexin 3, for example, expresses over 1,700 protein variants from splicing of one pre-mRNA sequence. Switches in splicing play major roles in almost all biological processes in complex organisms, including differentiation and the development of tissues and organs, apoptosis, senescence and ageing, long-lasting memory responses and direct responses to signals, such as caffeine or thermal control of diurnal rhythms in Drosophila and mice. Splicing switches are therefore deeply embedded determinants in biological processes, with the corollary that they are therefore also often involved in mediating or causing disease, including all forms of cancer.

Splicing events are controlled by numerous RNA-binding proteins that bind to the pre-mRNA. These proteins often bind promiscuously and weakly. Some act as repressors, others as activators, and many can be either of these depending on their sites of binding. A further complication arises because many have intrinsically disordered regions that can mediate interactions or phase effects. The mechanisms by which they affect splicing are largely still unclear. We are using single molecule microscopy, single molecule mass specrometry, structural biology and chemical and biological probes of flexibility to work out the mechanisms of control. The work is a collaboration between biochemists, structural biologists and physical chemists in Leicester, all in the Leicester Institute for Structural and Chemical Biology, and colleagues in synthetic chemistry in Strathclyde (Prof. G. A. Burley) and nano-engineering in Glasgow (Dr A. Clark), and it involves using state-of-the-art methods and developing the new methods required for investigating complex systems.

This project will involve working with the multi-disciplinary team. It is concerned with developing methods for following events in real time, both in vitro and in cells. It will lead to following single molecules of labelled protein by fluorescence microscopy, and analysing the dynamics of movement and interactions. Apart from microscopy, it will involve cloning and other standard molecular biology methods, in vitro assays of splicing and complex formation, and protein purification and characterization. It will lead to a PhD in Biochemistry.

Successful applicants will have a relevant first degree at a level equivalent to an upper second class, or higher, and some knowledge of chemistry, maths and computer programming.