The long term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism by which Dscam alternative splicing is mutually exclusive. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33, and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. Dscam is therefore the most extensively alternatively spliced gene known. The Dscam proteins functions as axon guidance receptors that play an important role in neural development and function. In addition, Dscam has been shown to function as immune receptors that help to defend the organism against pathogens. Current evidence suggests that the identity of the isoforms expressed in individual neurons is critical for proper wiring of the nervous system and in hemocytes is important for pathogen recognition. Thus, understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neuronal wiring and pathogen recognition in Drosophila. This proposal is aimed at understanding the mechanisms involved in regulating alternative splicing and the mechanism of mutually exclusive splicing of Dscam. First, we will determine the expression pattern of the exon 4 variants in the nervous system at single cell resolution and explore how regulatory proteins we have identified in tissue culture-based RNAi screens control Dscam splicing in individual neurons in the fly. Second, we will address many important issues regarding the mechanisms of mutually exclusive splicing. Specifically, we will functionally dissect the role of RNA secondary structures in exon 6 mutually exclusive splicing and identify other features such as distance, splice site strength, and HRP36 binding impact exon 6 splicing. Moreover, we will determine the order in which the Dscam introns are removed from the pre-mRNA as this has important implications on the splicing regulatory mechanisms. We will also determine if the splicing of exons within one cluster impacts splicing in other clusters. Finally, we will compare the ability of the insect and vertebrate splicing machinery to process pre-mRNAs containing clusters of three or more mutually exclusive exons.

Public Health Relevance

These experiments will provide tremendous insight into the mechanisms of alternative splicing and mutually exclusive splicing. As the vast majority of human genes are alternatively spliced, it is likely the discoveries we make will be of direct relevance to human health. Moreover, the results will provide significant insight into the mechanisms of neural wiring and immunity.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Connecticut
Schools of Medicine
United States
Zip Code
Plocik, Alex M; Graveley, Brenton R (2013) New insights from existing sequence data: generating breakthroughs without a pipette. Mol Cell 49:605-17
Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex M et al. (2013) Dynamic integration of splicing within gene regulatory pathways. Cell 152:1252-69
Bolisetty, Mohan T; Graveley, Brenton R (2013) Circuitous route to transcription regulation. Mol Cell 51:705-6
May, Gemma E; Olson, Sara; McManus, C Joel et al. (2011) Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster. RNA 17:222-9
McManus, C Joel; Graveley, Brenton R (2011) RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 21:373-9
Lee, Christopher; Kim, Namshin; Roy, Meenakshi et al. (2010) Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution. RNA 16:91-105
Nilsen, Timothy W; Graveley, Brenton R (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463:457-63
Graveley, Brenton R (2008) Molecular biology: power sequencing. Nature 453:1197-8
Graveley, Brenton R (2008) The haplo-spliceo-transcriptome: common variations in alternative splicing in the human population. Trends Genet 24:5-7
Olson, Sara; Blanchette, Marco; Park, Jung et al. (2007) A regulator of Dscam mutually exclusive splicing fidelity. Nat Struct Mol Biol :

Showing the most recent 10 out of 23 publications