C. elegans engages in three types of nuclear pre-mRNA processing: normal cis-splicing and trans-splicing of two different leaders: SL1 near the 5' end of pre- mRNAs, and SL2 at internal trans-splice sites in polycistronic pre-mRNAs to divide then into gene-length mRNAs. These three processes are mechanistically closely related,yet functionally distinct. How do C. elegans splicesomes pair 5' splice located in cis with an intron 3' splice site, but utilize only the appropriate SL for the two types of trans-splice site? It is argued that the highly conserved extended consensus, UUUCAG, at the 3' splice site is the key sequence for initiating splicing. Single base changes in this sequence will be tested for alterations in splice site choice in vivo. By analogy with mammalian splicing, it seems likely that U2F is the molecular responsible for recognition of this sequence. The gene for the C. elegans U2AF large subunit, which binds RNA and is required for splicing, has been cloned and recent results indicate it is alternatively spliced. Characterization of the U2F gene will be completed, the protein or proteins will be expressed, and antibodies obtained. The small subunit gene will also be cloned. Their map locations will be determined, and mutations that alter or destroy U2AF function will be selected. U2AF made in E. coli will be tested for binding specifically to the UUUCAG sequence using gel mobility, shift competitions and SELEX assays. Locations of a gene in a downstream position in a polycistronic transcription unit (operon) is sufficient to cause trans-splicing of its mRNA with SL2. The major focus of this project is to understand the mechanism of SL2 specific trans-splicing at internal sites in operons. A recently-developed in vitro trans-splicing system from C. elegans embryo extracts will be used to study how SL2 is specified. Polycistronic transcripts will be tested for specificity of trans- splicing in vitro. Trans-splicing specificity will also be investigated in transgenic animals, by altering gene spacing, the 5' cap, the nearby poly(a) signals, and the intercistronic sequence. The location of SL2 RNA where the specificity determinants reside will also be studied. Sequence required for 3' end formation, including the GU box and transcription termination signals will be determined. Differences between 3' end formation within an operon and at the 3' ends of transcription units will be investigated. New operons will continue to be identified to determine whether they involve co-expression of genes whose product function together, and to learn how much variation in operon structure is tolerated. The evolution and function of operons will be studied by a search for existence of polycistronic units in other genera. Molecules involved in trans-splicing will be identified by both genetic and biochemical techniques. Mutants that have lost the capacity for SL1 and SL2 trans-splicing at restrictive temperature will be selected. Proteins that interact with U2AF, or with SL1 or SL2 RNA's will be identified by in vitro binding experiments. In addition, the effect of overexpression of U2AF subunits will be studied, and dominant negative mutants will be sought by overexpression of U2AF missing RNA- binding or subunit interaction domain.
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