The long term objectives of this application are to understand in more detail two crucial aspects of RNA processing. These are i) the earliest step of spliceosome assembly and ii) how mRNA is exported from the nucleus to the cytoplasm. Many human diseases are due to aberrant splicing. Normal splicing, which is similar in yeast and mammals, precisely removes intervening sequences and joins together the exonic regions. The splicing process begins with formation of the U1 snRNP-pre-mRNA complex, the commitment complex in yeast or the E complex in mammals. In both systems, this process leads to functional pairing of the 5' and 3' splice sites. This early step of splicing has recently been shown to be critical for programmed cell death in mammals, indicating that it is important in a wide range of human diseases such as cancer. It is also involved in dosage compensation in fruit flies and meiosis regulation in yeast, suggesting that it is both a highly conserved process and amenable to study in yeast. mRNA export is less well understood, but the same genes and proteins are implicated in yeast and mammals. This proposal will define new genes involved in these processes as well as their biochemical mechanisms. This will involve the development of a new genetic method, which will also be applied to mammalian cell culture systems. The biochemical work will address structural and mechanistic aspects of commitment complex formation as well as define further the control steps required for mRNA export. Because of the genetic emphasis, these RNA processing studies will be done primarily in the yeast, Saccharomyces cerevisiae.
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