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. In collaboration with Andrew Hudson (Leicester), Cyril Dominguez (Leicester) and Glenn Burtley (Strathclyde), we are investigating the mechanisms of action of proteins that regulate splicing. Andrew Hudson, Cyril Dominguez an Ian Eperon are all members of LISCB, the Leicester Institute for Structural and Chemical Biology.

RECENT HIGHLIGHTS

SRSF1 (Jobbins et al., 2018), in collaboration with Glenn Burley (Strathclyde) and Andrew Hudson (Leicester)

We have recently published an analysis of the way in which the splicing factor SRSF1 engages with enhancer sequences when it stimulates splicing. This was awarded 'Breakthrough' status by Nucleic Acids Research, and the text of the notice is reproduced here.

A collaborative research team led by Drs. Glenn Burley, Andrew Hudson and Ian Eperon at the University of Strathclyde and University of Leicester (UK) has described studies that provide important new insight into the mechanisms that regulate pre-mRNA splicing in mammalian cells.

Exonic splicing enhancers (ESEs) are sequences that play key roles in determining whether the exons in which they reside are incorporated into the mature mRNA.  They were discovered and characterized in the mid-1990's, when it was demonstrated that canonical ESEs are bound by SR proteins (the prototype of which is SRSF1) to activate particular splicing patterns. Based largely on elegant experiments with Drosophila enhancers performed 20 years ago by the Maniatis lab, this process is generally thought to involve stable binding of the SR protein to an ESE, followed by rate-limiting interactions with factors bound at nearby splice sites.  However, it is difficult to reconcile this model with the recently discovered density of regulatory sequences in mammalian exons, and there have been no molecular tests that might resolve this discrepancy.

In this study, the investigators employ single molecule multi-colour colocalization to quantify stable SRSF1 binding in a nuclear extract that supports splicing, and chemical biology to dissect the subsequent activation process.  The results demonstrate that SRSF1-dependent mammalian enhancers do not follow the Drosophila paradigm, thereby breaking with traditional views of ESE function. Specifically, the authors conclude that the initial binding of SRSF1 is weak, and that interactions involving 3D diffusion incorporate a single molecule of SRSF1.  The picture that emerges is one in which exons do not form stable complexes with SR proteins that inevitably lead to splice site usage.  Instead, there appears to be a dynamic interplay among regulatory proteins that bind only transiently to the RNA, and the outcome depends on the chance that an SR protein is occupying an ESE at a particular moment, such as when the ESE is close to the 3’ splice site.

These findings require a profound change in the way in which the mechanisms of alternative splicing are studied.  Hitherto, both in vitro and in vivo, the emphasis has been on ‘what’ and ‘where’:  which regulatory proteins bind where on the pre-mRNA. Now, however, ‘when’ will have to be added:  it will be important to find the probability of binding, the lifetime of the occupied state, the numbers and combinations of proteins bound concurrently and the probabilities of interactions with target splice sites.  Moreover, in vivo, this dynamic flux is superimposed on the ways in which transcription itself affects the times of binding of regulatory factors, as first proposed by Eperon thirty years ago.  Addressing these challenges is a formidable task for the years to come.

Reviewers and editors familiar with the work have stated that the study "re-addresses long-standing open questions about ESE action using approaches that would have been impossible twenty years ago ", that the results are "unexpected and contrast with the interpretation of (historical) data" and that the work "provides interesting new insights that could not have been obtained by conventional ensemble methods and that will be of wide interest in the field of splicing regulation and its mechanisms."

The University of Strathclyde is the second oldest university in Glasgow, Scotland and one of the UK's first technological universities by Royal Charter.  The University of Leicester is a public university founded in the early 20th century that has recently created the Leicester Institute for Structural and Chemical Biology, to which Hudson and Eperon belong.  The Burley, Hudson and Eperon laboratories study the chemistry, biology and mechanism of regulated RNA splicing, among other topics.  The research project was supported by the Leverhulme Trust.

Nucleic Acids Research is a publication of Oxford University Press and is fully open access.  Its mission is to provide outstanding, scientist-led evaluation and dissemination of the highest caliber research across a wide range of disciplines focused on the role of nucleic acids and nucleic acid interacting molecules in cellular and molecular biology.  Breakthrough articles at NAR describe studies that solve a long-standing problem in their field, or provide exceptional new insight and understanding into an area of research that will clearly motivate and guide new research opportunities and directions.  They represent the top papers that NAR receives for publication, and are selected by the Editors based on nominations by authors and/or reviewers, and on the subsequent recommendation of the reviewers and editorial board members.

"The mechanisms of a mammalian splicing enhancer"

(Andrew M. Jobbins, Linus Reichenbach, Christian M. Lucas, Andrew J. Hudson, Glenn Burley and Ian C. Eperon; University of Strathclyde and University of Leicester)

ABSTRACT:

"Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors.  It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets.  However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise.  Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE.  We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3’ splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3’ splice site.  These insertions did not block activation, and we conclude that the activation step involves direct interactions.  These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site."

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 .


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 were involved in the first experiments to show that the splicing of an endogenous dystrophin gene could be manipulated for therapeutic purposes, in collaboration with George Dickson, and developed a method in collaboration with Francesco Muntoni, in ICH, UCL, to rescue the splicing of an exon in spinal muscular atrophy. Currently, we are collaborating with Cyril Dominguez (Leicester) and Glenn Burley (Strathclyde) to investigate whether G-quadruplexes affect the splicing of genes that either inhibit or promote apoptosis, with a view to finding new ways to shift cancer cells towards cell death.

 

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Redfearn Lecture 2017

To Be Confirmed