Although it might he logical to assume that, at some molecular level all lower and higher eukaryotes share a common mechanism for pre-mRNA splicing, a variety of in vivo and in vitro gene transfer experiments indicate that critical differences exist between the mammalian, yeast and plant RNA processing events. These differences, which may result from variations in the sequences recognized by intron splicing components, extend to the monocot and dicot groups of plants and have prevented the efficient in vivo expression of monocotyledonous genes containing introns in dicotyledonous plants. Although these processing deficiencies exist, no intron processing studies have been initiated to determine the critical differences between monocot and dicot pre- mRNAs which prevent the efficient excision of monocot introns in dicot nuclei. The experiments proposed herein are aimed at determining the RNA processing intermediates formed during excision of plant introns and at determining the fundamental differences between monocot and dicot RNA processing signals which block the expression of plant genes in heterologous nuclei. Specifically the research described in this proposal is aimed at more clearly defining the molecular parameters that determine the accuracy and efficiency of RNA processing in plant cells. The detailed objectives of this project are: (1) To identify and characterize the nucleotide sequences within dicot pre-mRNAs that are required for accurate and efficient excision of introns in dicot nuclei; (2) to identify the snRNA and protein components of dicot nuclear extracts responsible for intron excision: (3) to evaluate the nucleotide sequences which prevent the efficient excision of monocot introns in dicot nuclei: (4) to develop monocot RNA processing extracts which can be used to evaluate the intron processing efficiency of monocot and dicot precursor transcripts.
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