Final Report Summary - CLIP (Mapping functional protein-RNA interactions to identify new targets for oligonucleotide-based therapy.)
After the RNA molecule is produced in the nucleus of eukaryotic cells, it needs to pass through diverse regulatory stages before it is ready to be used as a template to produce a protein. One of the crucial stages is alternative splicing, where exons of a gene are combined in multiple possible ways. Alternative splicing produces multiple mRNA isoforms from a single gene, which can result in production of proteins with different functions. RNA-binding proteins play a crucial role in regulating the inclusion of alternative exons. The primary focus of this ERC project was to understand on a global scale how proteins regulate alternative splicing. Moreover, we aimed to provide insights into the role of alternative splicing in neuronal function and disease.
In order to understand the regulation of alternative splicing, we mapped protein-RNA interactions in a genome-wide manner. We developed a method that employs ultraviolet light to crosslink proteins to RNA in live cells, and then uses high-throughput sequencing to identify the crosslink sites with single nucleotide precision. Using this method, we characterised the function of hnRNP C, one of the most abundant proteins in cell nucleus. We identified over thousand exons regulated by hnRNP C, and found that hnRNP C silences inclusion of alternative exons when bound at the 3’ splice site. We also showed that the opposite rule applies to TIA proteins, which enhance exon inclusion when binding at 5’ splice sites. These are two examples of the several proteins we studied, which together led us to the finding that RNA-binding proteins find general position-dependent rules to regulate alternative splicing. We summarised these rules in the form of ‘RNA splicing maps’.
To understand how alternative splicing contributes to neurologic diseases, we addressed the function of two RNA-binding proteins, TDP-43 and FUS. Mutations in the genes encoding these proteins can cause motor neurone disease. We defined the in vivo binding sites of these proteins, studied their role in alternative splicing, and identified their mRNA targets in healthy and diseased human brain. We found that they regulate mRNAs encoding proteins that function in neuronal development or are implicated in neurological diseases. Moreover, we analysed the splicing changes in aging and neurodegeneration, which identified a network of splicing changes that may represent a link between aging and neurodegeneration.
Taken together, we showed that RNA-binding proteins can control alternative splicing in diverse manners, following positional principles that can be described by RNA maps of protein-RNA interactions. We assessed how RNA processing contributes to the diverse functions and changes that occur in brain during aging or disease.
In order to understand the regulation of alternative splicing, we mapped protein-RNA interactions in a genome-wide manner. We developed a method that employs ultraviolet light to crosslink proteins to RNA in live cells, and then uses high-throughput sequencing to identify the crosslink sites with single nucleotide precision. Using this method, we characterised the function of hnRNP C, one of the most abundant proteins in cell nucleus. We identified over thousand exons regulated by hnRNP C, and found that hnRNP C silences inclusion of alternative exons when bound at the 3’ splice site. We also showed that the opposite rule applies to TIA proteins, which enhance exon inclusion when binding at 5’ splice sites. These are two examples of the several proteins we studied, which together led us to the finding that RNA-binding proteins find general position-dependent rules to regulate alternative splicing. We summarised these rules in the form of ‘RNA splicing maps’.
To understand how alternative splicing contributes to neurologic diseases, we addressed the function of two RNA-binding proteins, TDP-43 and FUS. Mutations in the genes encoding these proteins can cause motor neurone disease. We defined the in vivo binding sites of these proteins, studied their role in alternative splicing, and identified their mRNA targets in healthy and diseased human brain. We found that they regulate mRNAs encoding proteins that function in neuronal development or are implicated in neurological diseases. Moreover, we analysed the splicing changes in aging and neurodegeneration, which identified a network of splicing changes that may represent a link between aging and neurodegeneration.
Taken together, we showed that RNA-binding proteins can control alternative splicing in diverse manners, following positional principles that can be described by RNA maps of protein-RNA interactions. We assessed how RNA processing contributes to the diverse functions and changes that occur in brain during aging or disease.