Principal Investigator Christopher Burge
The RNA splicing machinery is directed to particular locations in RNA transcripts by exon and intron sequences that either enhance or silence splicing at nearby splice sites. We have developed computational approaches to identify splicing enhancer elements that are active in mammalian cells, and found that these elements are predictive of splicing phenotypes of mutations in human genes. We are exploring the evolution of splicing enhancers to understand the extent to which different classes of enhancers have distinct or interchangeable functions.
To discover silencers of splicing, we have developed a cell fluorescence-based screening method, and used this approach to identify a diverse collection of exonic splicing silencer (ESS) motifs. Almost all of these elements share a consistent pattern of context-dependent activity, silencing splicing when present in exons, activating splicing when present in introns, and altering splice site choice when present between competing splice sites. We are exploring the roles that ESSs play in a variety of types of regulated alternative splicing events, using comparative genomic and molecular approaches, and integrating ESS and other motifs into algorithms that simulate exon recognition by the splicing machinery. Reverse genetics (RNAi) is being used to identify splicing factors required for the activity of these ESS motifs.
Core splice site motifs and splicing regulatory elements function together to regulate splicing decisions. We are beginning to explore the functional interactions between classes of splicing regulatory elements, using genomic/comparative genomic analyses together with splicing reporter assays. One direction involves identifying pairs of elements that preferentially occur together in adjacent gene regions. This approach has led to the identification of a pair of intronic motifs that can function cooperatively to silence the splicing of intervening exons. Pairs of co-occurring motifs in other gene regions are also being explored. Patterns of evolutionary compensation between different classes of splicing regulatory elements can also provide clues to functional interactions. We are exploring in depth a pattern in which intronic splicing enhancers (ISEs) enhance the splicing of exons whose 5' splice sites have moderate intrinsic strength much more strongly than for other exons whose splice sites are either weaker or stronger. This splice site strength-specific activity suggests unexpected complexity in the activity of splicing enhancers. To explore the regulatory functions of alternative splicing in mammalian differentiation, in collaboration with the Phillip Sharp lab we are using splicing-sensitive microarrays to analyze changes in the expression of alternative mRNA isoforms following activation of primary T-cells.