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Detection in cells of protein interactions on pre-mRNA during the regulation of splicing

Primary Supervisor: Professor Ian Eperon, Department of Molecular and Cell Biology

Secondary Supervisor: Professor Shaun Cowley, Department of Molecular and Cell Biology

PhD project title: Detection in cells of protein interactions on pre-mRNA during the regulation of splicing

University of Registration: Univeristy of Leicester

Project outline:

One of the biggest challenges in molecular biology is presented by the selection of sites for RNA splicing. We cannot overstate its importance or apparent complexity. Splicing determines which mRNA and protein sequences a gene expresses. It is accurate, removing introns of 102-106 bases, but paradoxically flexible in mammals, where most genes produce multiple isoforms of mRNA. These may produce proteins with different functions (up to 1,800 functional isoforms of neurexin 3, for example) and switching is involved in memory, development, differentiation, signalling and disease. Numerous regulatory proteins and their binding sites have been identified by ensemble and ‘omic’ approaches, but our understanding of their mechanisms and functional integration remains very poor. We have recently made breakthrough findings by combining single molecule methods and chemical biology. These methods and a transformative vision of the dynamics of the process leave us poised to discover the mechanisms of selection.

This research is based on a multi-disciplinary group from the Universities of Leicester, Strathclyde and Glasgow. This group brings together expertise in nano-engineering, bio-organic chemistry, photonics, structural biology, RNA splicing and molecular biology, and has been funded by a BBSRC sLOLA grant for 5 years. This PhD project is concerned with in vivo tests of the predictions from our in vitro results, and therefore provides a very important complement to the more biophysical work. It will involve the use of RNAi and over-expression, mutations and CLIP to test protein interactions in cells. More direct evidence will come from biotin proximity ligation assays, in vivo protein-protein interaction assays based on fluorescence microscopy and novel methods for targeting protein cross-linking to proteins on specific pre-mRNA substrates.

The University if Leicester is very well equipped for this research, with extensive conventional cell microscopy resources and expertise in cell and molecular biology in the supervisors’ labs.

See: Jobbins, A.M., Reichenbach, L.F., Lucas, C.M., Hudson, A.J., Burley, G.A., & Eperon, I.C.* (2018). The mechanisms of a mammalian splicing enhancer. Nucleic Acids Research 46, 2145-2158 (doi: 10.1093/nar/gky056). Breakthrough article.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology 

Techniques that will be undertaken during the project:

  • Molecular biology
  • In vitro transcription and splicing
  • Expression of transfected genes and gene silencing
  • Analysis of RNA-seq and mass spectrometry
  • Mutagenesis, analyses of complexes
  • Fluorescence microscopy and in vitro assays of protein binding to RNA

Contact: Professor Ian Eperon, University of Leicester