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

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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 .