| Description |
The field of RNA editing was discovered when non-genomic information, base transitions, transversions, insertions or deletions, were found in tRNA, rRNA and mRNA sequences. RNA editing by adenosine deaminases that act on RNA (ADAR) convert adenosine (A) to inosine (I) in double-stranded RNA. ADARs are present in all metazoans, and the two ADARs from C. elegans, adr-1 and adr-2, are characterized by molecular, biochemical and genetic techniques in this dissertation. First, the cloning of adr-1 splicing variants and the neural expression pattern of adr-1 are described. Homozygous knockouts of adr-1, adr-2, or both adrs are characterized in vitro and in vivo for ADAR activity. C. elegans lacking adr-1 have greatly diminished RNA editing, and those lacking adr-2 have no detectable A-to-I editing. A chemotaxis defect phenotype is shown to be more severe in strains that lack all RNA editing by ADARs, adr-2(-l-) and adr-1 (-/-);adr-2(-/-), than for the adr-l{-l-) strain alone. A normal chemotaxis response is restored by introducing a wild-type copy of adr-2 into adr-2{-/-) strains (Chapter 2). Significantly, the chemotaxis defect of adr{-l-) strains was rescued by mutations in the RNA interference (RNAi) pathway. ADARs were previously known to modify protein function via editing of codons to specify different amino acids or create new splice sites. Neurological defects have been linked to loss of A-to-I editing at a single codon site, and the C. elegans chemotaxis defect was predicted to also involve a codon change mediated by ADARs. The rescue of chemotaxis by mutations in the RNAi pathway defines a previously unknown function for ADARs in keeping transcripts with double-stranded regions from entering the RNAi pathway (Chapter 3). Finally, a microarray experiment comparing wild-type and adr mutant C. elegans was performed. However, no significantly altered transcript levels were identified in adr mutants aside from the deleted gene adr-2. As the incomplete chemotaxis phenotype suggests, changes in transcript level may be subtle and require more creative methods to enable detection by microarray. |
