Award details

Understanding pre-mRNA splicing regulation with novel inhibitors

Reference	BB/S00047X/1
Principal Investigator / Supervisor	Professor Raymond O'Keefe
Co-Investigators / Co-Supervisors	Dr Richard Bryce, Dr Roger Whitehead
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	604,841
Status	Completed
Type	Research Grant
Start date	01/01/2019
End date	30/04/2022
Duration	40 months

Abstract

Pre-mRNA splicing is essential for gene expression during development, differentiation, environmental responses and aging. Misregulation of splicing is associated with numerous diseases. Splicing removes introns with single nucleotide precision producing mRNA for protein synthesis. Splicing is catalyzed by a large RNA/protein complex called the spliceosome that is assembled with a pre-mRNA, activated for intron removal, then disassembled to allow subsequent splicing rounds. The splicing activation and disassembly steps are facilitated by the ATPase Brr2 whose activity is regulated by the GTPase Snu114. Brr2 unwinds the U4/U6 and U2/U6 snRNAs required for spliceosome activation and disassembly, respectively. We have now identified two small molecules, fusidic acid and lithocholic acid, which specifically inhibit both yeast and human pre-mRNA splicing. These molecules target the core spliceosome protein Snu114 which regulates spliceosome activation and disassembly through Brr2. The exact mechanisms by which Snu114 regulates Brr2 function during spliceosome activation and disassembly are unknown. We propose to use molecular modelling and synthetic chemical approaches to develop more potent and specific analogues of fusidic acid and lithocholic acid which will be important tools to investigate Snu114 function. We will then specifically investigate whether Snu114 regulates Brr2 by allowing it access to its snRNA substrates, whether Snu114 directly activates Brr2 RNA unwinding activity and how inhibition of Snu114 function influences the global splicing programme in cells. The proposed work will combine chemistry, computer modelling, biochemical, genetic and next generation sequencing approaches in the yeast and human systems to provide a comprehensive analysis of Snu114 function in regulating pre-mRNA splicing. As the regulation of pre-mRNA splicing is essential for gene expression this work will have wide interest and impact.

Summary

Genes within cells are copied into a pre-messenger RNA (pre-mRNA) which is used as a template for protein production. All information contained within genes is not required for making proteins. Unwanted information, therefore, must be removed from the pre-mRNA before it is used for protein production. Unwanted information is removed, or spliced, from pre-mRNA through a process similar to the editing of unwanted frames from a film. One end of the region to be removed is first cut then the other end is cut removing the unwanted region, while the two remaining pieces are spliced together. This splicing of pre-mRNA is important because it must occur very accurately in order for functional proteins to be produced. Splicing is essential for all aspects of human biology including proper embryo development and the formation of all tissues and organs. Splicing is also vital for organisms to respond to their environment and adapt to stresses and nutrient deprivation. Defects in splicing are associated with a wide range of diseases including developmental disorders, diabetes, cancer and age related diseases. Alternative splicing, which allows different portions of genes to be put together in many different combinations, has also allowed humans to expand their cellular complexity without having to increase the size of their genome. The work that will be undertaken here will utilize novel chemical tools to address how the process of splicing occurs as there are still some key unanswered questions on how splicing takes place and how it malfunctions in certain diseases. Splicing is carried out by a large RNA/protein complex called the spliceosome. The spliceosome arranges itself into specific conformations to identify and "splice" out the unwanted regions. The spliceosome must be assembled on to pre-mRNA, activated to allow splicing to occur and then disassembled to allow subsequent rounds of splicing. To date there is still much we do not know about the assembly, activation anddisassembly of the spliceosome. Small molecule inhibitors of pre-mRNA splicing that specifically block certain steps of spliceosome assembly, activation and disassembly would be valuable tools for deciphering spliceosome function. Small molecules have proven invaluable for determining the function of other RNA/protein complexes like the ribosome which is responsible for making proteins in the cell. Unfortunately, there are very few small molecule inhibitors of splicing, therefore the development of new small molecule inhibitors of splicing would greatly enhance research in this field. In work leading up to this proposal we have identified two new small molecule inhibitors of splicing, fusidic acid and lithocholic acid. We believe these two molecules target an important protein in the spliceosome, Snu114, that is known to regulate both spliceosome activation and disassembly. Developing these two new small molecules as inhibitors of splicing will now allow scientists to understand the mechanisms of splicing in more detail. As we are the first researchers to discover the effects of these molecules on splicing, we now have an advantage in further developing these molecules for the analysis of splicing mechanisms. Work proposed during the tenure of this research grant will use molecular modelling and synthetic chemistry approaches to develop improved analogues of fusidic acid and lithocholic acid. These novel analogs will then be used in combination with other experimental tools to investigate the mechanisms of pre-mRNA splicing and how splicing is regulated in both yeast and humans. Pre-mRNA splicing is affected in many diseases and basic research into approaches that modulate splicing have already led to effective therapies, like that recently seen for Spinal Muscular Atrophy. Therefore, this research will provide information on the basic mechanisms of pre-mRNA splicing that can inform research into therapies for diseases that impact pre-mRNA splicing.

Impact Summary

Who will benefit from this research? We have identified numerous groups of users and beneficiaries outside the academic research community who will benefit from the research in this proposal. These are clinical researchers, industrial collaborators and third-sector organizations. We are also actively engaged in activities that increase the public understanding of science through visits to schools and demonstrations at the Manchester Museum. How will they benefit from this research? The research in this proposal is basic research. The knowledge obtained through this research will provide the fundamental theories and concepts underlying cell function, gene expression, development and disease. We can impart this new knowledge to our student and public beneficiaries through the numerous engagement activities we undertake (see Pathways to Impact). In addition, the fundamental theories and concepts we discover will provide information for more disease-oriented investigations by clinical researchers. Our research into the regulation of RNA splicing may also benefit commercial private sector researchers who are current collaborators. Specifically, the small molecule inhibitors we develop may be useful in modulating splicing activity in certain disease states which could be developed by our commercial collaborators. Third sector organizations such as Genetic Alliance UK, Genetic Disorders UK, Rare Disease UK, Sparks and Newlife Foundation, who are interested in causes/treatments of rare genetic diseases, will also be interested in the results of our research. What will be done to ensure that they have the opportunity to benefit from this research? Our labs have engaged with primary school children through presentations about DNA and genetics at a local school. We have engaged with secondary school children through the "Researchers in Residence" programme, through presentations at Manchester Museum, through workshops at NOWGEN Centre for Genetics in Healthcare and through writing articles for the "Biological Sciences Review". We have engaged University students by discussing and presenting our work through practical and lecture courses at our University. All these engagement activities will continue and develop through feedback from the beneficiaries. We will directly contact clinical researchers, third sector organisations, our current industrial collaborators and newly identified industrial links to inform them of our research. We will work closely with the University of Manchester Intellectual Property (UMIP) to investigate commercial options resulting from research projects and negotiating intellectual property rights.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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