Ian Eperon's Research Interests

Research into the mechanisms of splicing, splice site selection, alternative splicing and therapies. We welcome enquiries from prospective PhD students interested in RNA splicing research, especially those with backgrounds in physics or physical chemistry. Contact Prof. Eperon directly by email.

Publications List

RNA splicing: single molecules to therapy

Ian Eperon

Almost all mammalian genes produce multiple isoforms of mRNA and protein by alternative splicing; this, not the number of genes, is the key to the evolution of complex eukaryotes. We are interested in the molecular mechanisms by which sites are selected and have made a number of contributions (see publications).

 

The very existence of extensive alternative splicing in mammals tells us something important: the splice site sequences are not very information-rich, and there are lots of candidate sites in the pre-mRNA. Some are used constitutively, some in specific circumstances, some only if the normal site is mutated, and others never. How is the RECOGNITION by specific factors turned into SELECTION of a site? Most nucleotides in or near some exons contribute to the use of an exon, presumably because they are bound by one or more of the many RNA-binding proteins that appear to compete for binding, with low and overlapping specificities. To understand recognition and selection, we need to understand what the pre-mRNA looks like: which proteins are bound, where, and in what numbers, and what effects they have on the behaviour of the RNA.

 

It became clear to me some years ago that splicing was too complicated for conventional molecular approaches to be very informative, and, in collaboration with Clive Bagshaw, we began a programme to develop single molecule methods for analysing splicing in nuclear extracts. Dmitry Cherny did the first single molecule experiments on mammalian splicing and showed that we could determine the numbers of regulatory proteins associated with each molecule of RNA in functional conditions (nuclear extracts). We are now in a position to address some of the most intractable problems.

RECENT HIGHLIGHTS

PTB (Cherny et al., 2010). In collaboration with Chris Smith and colleagues, Cambridge.

  • Established for the first time the numbers of molecules of a regulatory protein bound to pre-mRNA
  • 5-6 molecules of PTB bind 2 regions flanking repressed exon 3 of TM1
  • Modelling with known domain structures revealed  new insights into the arrangements of proteins on the RNA, and in particular suggested that for proteins with multiple RNA-binding domains, with similar RNA sequence specificities, the sites with the highest apparent affinity are those that enable the highest number of possible arrangements of the domains on the RNA .


5' SS SELECTION BY U1 snRNPs (Hodson et al., 2012)

  • Single molecule methods showed that in early complex E two U1 snRNPs bind pre-mRNA with 2 strong 5' splice sites.
  • This suggests U1 snRNPs bind independently and stochastically to alternative sites, not via recruitment of a single U1 snRNP as would be expected on the basis of existing models for complex E.
  • In complex A, only one is bound; there is a process associated with ATP-dependent complex A formation in which the surplus U1 is removed. This is linked to commitment to a specific 5' SS.
  • The results suggest models for selection of weak and strong sites:
  • The affinity-dependent selection among weak sites arises because the probability of a site being occupied by U1 is low and reflects the affinity.
  • The position effect (favouring the intron-proximal site) arises when there are alternative strong sites because both are occupied concomitantly.
  • Why is the intron-proximal site favoured? Calculations suggests that this is not the result of free diffusion of an RNA chain. A better match to our 1991 data on the effects of distance between the sites is seen if we model the exon as a RIGID rod. Does U1 or protein binding change the physical properties of an exon?


AN ENHANCER THAT DOES NOT ACT BY LOOPING (Lewis et al., 2012). This is a joint project with Glenn Burley and colleagues, Strathclyde.

  • The general model for the action of exonic splcing enhancers is that they act by looping of a free RNA chain (just as was expected for the selection of 5' splice sites when there is simultaneous occupancy by U1 snRNPs), but there was little evidence.
  • We placed an enhancer upstream of two alternative 5' splice sites; it shifted splicing to the nearest site.
  • We inserted a non-RNA flexible linker between the enhancer and the sites, using click chemistry. This abolished the effect of the enhancer.
  • The conclusion is that the enhancer must in some way exert its effects along the connecting RNA. As with the U1 work, does this suggest that protein complexes propagate along the exon from an enhancer (and make it more rigid, perhaps?).

 

We are developing the power of single molecule methods further in collaboration with Andrew Hudson (Chemistry, Leicester) to observe single molecule reactions in real time in a laser trap.

Defective or altered splice site selection is the cause of many genetic diseases and is required for diseases such as cancer. We developed a method some years ago, in collaboration with Francesco Muntoni, in ICH, UCL, to rescue the splicing of an exon in spinal muscular atrophy. We are optimizing this, trying to understand the reasons for its success, and applying the method to genes involved in cancer to switch splicing either to favour cell death (in collaboration with Cyril Dominguez, Leicester) or prevent invasiveness (collaboration with Guiseppe Biamonti, Pavia).

 

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Department of Molecular and Cell Biology
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Redfearn Lecture 2017

To Be Confirmed