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Use of single molecule fluorescence and cryo-EM to study the regulation of RNA splicing

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

Secondary Supervisor: Dr Andrew Hudson, Department of Chemisty

PhD project title: Use of single molecule fluorescence and cryo-EM to study the regulation of RNA splicing

University of Registration: University 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. The PhD project will fit into the heart of this consortium. It will involve the analysis of splicing regulatory proteins, fitted with fluorescent tags, and their association with pre-mRNA and with other proteins in individual complexes. In addition, it will involve developing novel methods for analysing complexes using cryo-electron microscopy. These approaches hold the possibility of making major advances in our understanding.

The University if Leicester is very well equipped for this research. We have several home-built single molecules microscopes, which we have been using for studying splicing for some years, and we have a relatively new and already highly productive cryo-electron microscope.

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 harvesting of nuclear extracts
  • Crosslinking assays
  • Single molecule fluorescence microscopy
  • Statistical analysis
  • Chemical derivatization of RNA
  • Conjugation of RNA
  • Purification and characterization of complexes
  • Electron microscopy
  • Cryo-electron microscopy

Contact: Professor Ian Eperon, University of Leicester