We propose to continue our studies on the structure and expression of the actin genes of the nematode Caenorhabditis elegans. We will now focus on actin messenger RNA (mRNA) processing and extend the work to include processing of other mRNAs. While studying the actin genes, we recently discovered that three of the four genes encode mRNAs that undergo an unusual reaction in which the 5' 22 nucleotides of the mRNAs are derived from a separate RNA molecule. Other mRNAs in C. elegans also undergo this trans-splicing reaction. We will study this phenomenon and its biological implications. We will elucidate the mechanism of trans-splicing of the actin mRNAs in C. elegans. We will characterize the actin mRNA precursors, the splice leader RNA precursor, and molecular intermediates. We will use in vitro splicing reactions to identify the components of the reaction, determine if separate trans-and cis- elements (snRNPs and spliceosomes) exist and cis-splicing is kept separate from trans-splicing we will identify other MRNAs that undergo the same transplicing reaction. We will investigate whether the splice leader confers mRNA stability, enhances translation, or localizes mRNAs. We will carry out experiments to determine whether similar splice leaders are acquired through trans-splicing in other eukaryotes.
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