Our long term goal is to understand early vertebrate development at the molecular level. We study the problem in the frog Xenopus, which produces large numbers of eggs that are readily manipulated by microinjection and microsurgery. A combination of experimental embryology and molecular xmanipulation provides methods to understand the roles of specific genes and signaling pathways in elaborating the structure of the embryo. Most of the paradigms for development of vertebrate embryos have come first from work with amphibians, and many of the signaling activities were first analyzed using amphibian embryos. Gain of function experiments using mRNA injection, and loss of function using Morpholino oligonucleotides have provided insights into the mechanisms that underlie tissue differentiation and morphogenesis. During previous grant periods, we have used expression cloning to identify potent signaling and signal transduction activities that contribute to embryonic development. In screens for embryonic activities that alter neural patterning we identified several RNA regulators that show specific effects in both gain of function and loss of function experiments. In the next grant period, we will analyze selected RNA binding and pre-mRNA splice regulating activities that show such highly specific effects on development, and will therefore gain new insight into the selective effect of these proteins on specific splicing choices. The control of alternative pre- mRNA splicing has emerged as an important mechanism in gene control, and recent methods allow a global analysis of changes in splicing that are directed by specific proteins. We will apply these methods in Xenopus to understand how these proteins regulate splice choices, and thereby resolve the previously unknown function of these splicing regulators in splicing choices.
Mapping of mutations that cause human diseases showed that many mutations are found in the conserved splicing junctions or branchpoint sequences of precursors to protein coding messenger RNAs, rather than in the protein coding sequence of the mRNA. It is therefore crucially important to understand the mechanisms that regulate the use of splicing signals, in order to understand the susceptibility to and etiology of diseases, as well as to devise therapies for such disease. This proposal will advance our understanding of splicing regulation using the model vertebrate organism, Xenopus, which affords technical advantages in manipulating splicing regulators, and studying the consequences of this manipulation.
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