Award details

Regulation of pre-mRNA splicing fidelity by the Nineteen Complex (NTC)

Reference	BB/I019510/1
Principal Investigator / Supervisor	Professor Raymond O'Keefe
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	314,220
Status	Completed
Type	Research Grant
Start date	01/01/2012
End date	31/12/2014
Duration	36 months

Abstract

Pre-mRNA splicing is essential for gene expression during development, differentiation, responses to the environment and ageing. Misregulation of splicing is associated with numerous diseases. Splicing removes introns with nucleotide precision producing mRNA for protein synthesis. Splicing is catalysed by a large RNA/protein complex called the spliceosome that assembles with a pre-mRNA, identifies the splice sites, then arranges into specific conformations for intron removal. The NineTeen Complex (NTC) associates with the spliceosome and is required for progression through splicing. Our recent published work proves that the NTC protein Cwc2 directly links the NTC to the spliceosome active site. Following this our preliminary data reveals that distinct mutations in Cwc2 either negatively influence the first step of splicing or reduce fidelity of the second step. We show using yeast genetic methods that Cwc2 is involved in forming and/or stabilising the U2/U6 helix I base-pairing conformation which contributes to the spliceosome active site. In addition, we have identified a Cwc2-pre-mRNA crosslink before the second step of splicing suggesting a role for Cwc2 in orienting the pre-mRNA for this step. We propose a new mechanism of spliceosome regulation to be tested here where the NTC is the target for spliceosomal ATPases that regulate conformation changes in the spliceosome. To confirm this new mechanism we will 1) determine the role of Cwc2 in establishing spliceosome conformations and the relationship of Cwc2 with ATPases known to regulate these conformations 2) elucidate the functional significance of Cwc2 interactions with the pre-mRNA and 3) investigate the contribution of protein phosphorylation in regulating Cwc2 function. Overall this work should provide information on how the NTC contributes to formation of spliceosome conformations during pre-mRNA splicing. As the NTC is highly conserved between all eukaryotes this work should be of 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 the information contained within genes is not required for making proteins. The unwanted information, therefore, must be removed from the pre-mRNA before it is used for protein production. The unwanted information is removed, or 'spliced', from pre-mRNA by 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 while the two remaining pieces are 'spliced' together. This 'splicing' of the pre-mRNA is very important because it must occur accurately in order for functional proteins to be produced. The regulation of splicing is essential for all aspects of human biology. Splicing is required for proper embryo development and differentiation of all tissues and organs. Regulation of pre-mRNA splicing is also vital for organisms to respond to their environment and adapt to stresses and nutrient deprivation. Defects in pre-mRNA splicing are associated with a wide range of diseases including diabetes, cancer and age related diseases. Alternative splicing 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 address how the process of splicing occurs as there are still some key unanswered questions on how pre-mRNA splicing is regulated. Splicing is carried out by a large RNA/protein complex called the spliceosome. The spliceosome must arrange itself into specific conformations to identify and 'splice' out the unwanted regions. An increasing amount of evidence points to a complex of proteins called the NineTeen Complex, or NTC, that associates with the spliceosome to act as an essential regulator of spliceosome function. In work leading up to this proposal we have identified an NTC protein called Cwc2 that provides the direct link between proteins of the NTC and the active site of the spliceosome. Discovering this link between the NTC and the spliceosome has implications for how scientists now understand the mechanisms of pre-mRNA splicing regulation. As we are the first researchers to discover this link we now have an advantage in discovering exactly how this link contributes to the regulation of pre-mRNA splicing. Work proposed during the tenure of this research grant will address how Cwc2 is involved in regulating spliceosome conformations required for the two steps of splicing through a set of detailed molecular experiments. As the basic mechanisms of pre-mRNA splicing are identical in yeast and human cells, we avoid using animals in our research by using yeast cells to perform our experiments .

Impact Summary

Who will benefit from this research? We have identified three groups of users and beneficiaries outside the academic research community that have benefited from the research in our lab in the past and who will continue to benefit from any research in the future. These are primary school children, secondary school children and University students. In the future we plan to engage with commercial private sector researchers. 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 and gene expression specifically. We can impart this new knowledge to our student 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 other researchers. Our research into the regulation of RNA splicing in yeast may also benefit commercial private sector researchers who are trying to use yeast as a system to produce protein and RNA products from yeast. What will be done to ensure that they have the opportunity to benefit from this research? Our lab has engaged with primary school children through presentations about DNA 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 work closely with the University of Manchester Intellectual Property (UMIP) which is a support service we have that includes investigating commercial options resulting from research projects and negotiating intellectual property rights.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology
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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