Trans-splicing involves the ligation of two separate RNA molecules into a single contiguous transcript. Trypanosomes, nematodes and trematodes carry out trans-splicing. In nematodes, the spliced leader RNA (SL RNA) transfers its 5'22 nucleotides from a consensus donor splice site to a consensus acceptor splice site located in the 5-untranslated region of a pre-mRNA. Consequently, some mRNAs carry a 22 nucleotide long 5'spliced leader sequence. Two proteins have been identified that bind specifically to mRNAs that contain an SL but fail to bind to mRNAs that lack an SL. The proposed research will define the detailed molecular interactions between these SL binding proteins and the SL nucleotide sequence. The methods include chemical modifications of the RNA and footprinting of the complex. Variant RNA molecules will also be selected that bind to the proteins with high affinity. These variants will help define a consensus RNA sequence and structure that will provide insight into the architecture of the molecular interactions. These studies will help establish the basic rules for RNA protein interactions. The biological function of the binding proteins and their interactions with SL on the mRNA will be studied genetically in C. elegans. The genes encoding the SL proteins will be identified. DNA sequence homologies with related genes will be sought that might relate the SL binding proteins to other RNA binding proteins of known function. The genes encoding the binding proteins will be disrupted by transposon mutagenesis. The phenotypes of those mutant nematodes lacking the binding proteins will be studied in order to define the null phenotype and to determine whether the proteins are essential. Mutations will be derived in the genes in order to elucidate their biological functions in the growth and development of C. elegans with particular attention to encoded protein domains of known functions, e.g. dominant negative phenotypes associated with nucleotide binding sites.
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