To elucidate the mechanisms that guide the assembly of thick filament proteins into the highly ordered contractile apparatus, we have undertaken an analysis of the
unc-82 gene. Mutations in
unc-82, which cause a mild uncoordinated phenotype, are associated with abnormalities in thick filament morphology and organization (Waterston et al., 1980). A combination of genetic mapping, transformation rescue, and RNAi experiments demonstrate that
unc-82 corresponds to predicted ORF B0496.3. Two transcripts, which differ by alternative inclusion of a single coding exon, encode ~1600 residue proteins that contain a serine/threonine kinase active site domain at the N-terminus. No other regions of homology to known proteins have been detected. BLAST analysis using the UNC-82 kinase domain has identified putative orthologs in Drosophila and vertebrates. Phylogenetic trees based upon comparisons of kinase domain sequences suggest that UNC-82 is a member of a novel class of kinases related to the SNF1 and PAR-1 kinase families. The
unc-82 phenotype suggests a role for the kinase in regulation of thick filament formation or function. The more severe allele,
e1323, contains a premature stop codon in the kinase domain, and is therefore likely to represent a null. The allele
e1220 contains a missense mutation (E202K) within a conserved residue of the catalytic loop. Inactivation of the locus by RNAi produces a phenotype similar to that of these alleles. Potential UNC-82 substrates include the homologous nonhelical tailpiece domains present in myosin and paramyosin (Schriefer and Waterston, 1989). Consistent with this possibility, a rescuing UNC-82::GFP fusion localizes to the M-line in body wall muscle. In current experiments, we are testing the in vivo role of the tailpiece in both myosin and paramyosin, and examining the ability of extracts from
unc-82 animals to phosphorylate endogenous paramyosin. The localization of UNC-82::GFP in nonmuscle cells suggests that UNC-82 may function in the organization of other cytoskeletal structures, such as the intermediate filament bundles in the marginal cells of the pharynx.