In the central nervous system, protein isoform diversity is responsible, at least in part, for its complexity and its many orchestrated activities. This proposal concerns the role of alternative splicing in the differentiation and function of various extracellular matrices (ECMs) of epithelial cells during the development of the model organism, Drosophila melanogaster. We will explore the possibility that different ECM protein isoforms, generated by both cis and trans alternative splicing of mRNAs encoded by the complex dumpy gene, characterize ECMs at the apical surfaces of epithelial cells in different tissues at different developmental stages. Subsequent investigations will determine if the isoforms resulting from alternative splicing are involved in various ECM functions such as adhesion, apoptosis, remodeling and signaling. Since Zona pellucida (ZP) domain proteins, like Dumpy, are important components of vertebrate ECMs, these studies will directly relate to certain human defects. Thus, mutations of the ZP domain protein, alpha-tectorin, lead to hearing loss and mutations in Endoglin are associated with a hemorrhagic vascular disease. Other ZP domain containing protein complexes are essential for fertility. An understanding of the structure and function of apical ECMs will shed light on these and other human diseases. Our approach will utilize a large collection of molecularly defined dumpy mutants which affect all or only a subset of Drosophila tissues and developmental stages. We will use primers located at strategic positions in the huge and complex dumpy gene for RT-PCR to detect cis alternative splicing events. Trans-splicing events will be examined in exons marked by complementing dumpy lethal nonsense mutations. Both the mutant sites themselves and single nucleotide differences (SNPs) will be used to detect recombinant transcripts.
Cells are held together in different tissues by means of the extracellular matrix (ECM). The ECM must be remodeled in the morphogenetic processes of development. We propose that, like the central nervous system, alternative splicing of ECM transcripts underlies its complexity, and will use the Drosophila dumpy gene to document this phenomenon.
| Carmon, Amber; Guertin, Michael J; Grushko, Olga et al. (2010) A molecular analysis of mutations at the complex dumpy locus in Drosophila melanogaster. PLoS One 5:e12319 |
