Grant: Operating Grant

Proteins are the workhorses of the cell and each protein is encoded by a gene. The human genome contains ~25000 genes, yet there are several hundred thousand different proteins. One gene must encode many proteins - how can this be? The answer is a process called splicing. Each gene is made up of blocks, or 'exons'. Splicing allows one gene to encode many proteins by using different combinations of exons. Imagine each protein is a word and that each exon of a gene encodes a letter. If a gene contained 8 exons, e.g. the letters S-P-L-I-C-I-N-G, then these letters can be used in different ways. They can be used to make full-length protein, SPLICING, or a shorter form like ICING. Crucially, more complex combinations like SPIN or SLICING can be made by skipping internal blocks. Each example here is a meaningful combination but there are other nonsensical possibilities like SPLG. How does the cell make only the meaningful splice forms and never the nonsensical ones? How do different tissues make different meaningful forms? This is what we want to examine. Surrounding each exon are signals that tell the cell whether to use the exon or not. These signals are seen by splicing factors but we do not know which factor sees which signal. Splicing in nematode worms is very like that in human but the genome is simpler and we can do direct experiments. We have already used cutting edge tools to find all the ways that exons are combined in normal worms. What we propose to do now is to remove each splicing factor in turn and see the effect on the pattern of splicing. In this way we will see which splicing factor is required for which splicing events. By examining the signals present around the affected exons, we can predict which factor sees which splicing signals. Finally, we confirm this by examining which signal each factor sees in a test-tube. Together, this will give us a deep understanding of how splicing is regulated, and how errors in splicing lead to human disease.