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A comprehensive RNAi screen for novel muscle-affecting genes in Caenorhabditis elegans identifies two paralogs, TO4A8.4 and F36F2.1, homologous to mammalian STARS Dube, Nicholas

Abstract

One of the fundamental features of metazoan development is myogenesis. A crucial step during myogenesis is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, four phenotypic classes of muscle mutants defective in some aspect of muscle structure and function have been identified in mutagenesis screens: the uncoordinated (unc) class, typified by uncoordinated, slow or no movement, the muscle positioning defective (mup) class, the late embryonic lethal class known as paralyzed and arrested at two-fold stage (pat) mutants, and the class where animals are capable of wildtype development and movement but have disorganized muscle (dim). Using SAGE and microarray chip analysis we have identified 3395 non-ribosomal/ non-mitochondrial genes expressed in muscle. Using an RNAi feeding library, we screened this 'muscle expressome' for genes affecting sarcomere assembly, stability and/or function. Worms harboring an extrachromosomal array containing a myosin heavy chain gene, myo-3, fused in frame to green fluorescent protein (GFP) were fed bacteria expressing dsRNA corresponding to each gene within the muscle expressome. The progeny of these worms were examined for both overt phenotypes and mislocalization of myo-3::GFP. This approach proved to be a rapid and sensitive means to identify genes required to organize sarcomeric proteins into a highly ordered myofilament lattice and we identified 296 genes with defects in myo-3.:GFP localization in an initial screen and reconfirmed 121 of those genes in a rescreen. RNAi treated animals display an array of myofilament disruptions ranging from small aggregations of myo-3::GFP to large deposits, often accompanied by disorganization of the myofilaments. The high percentage of tested genes affecting muscle sarcomeres, 3.6%, likely reflects the fact that we have already enriched for genes expressed in this tissue. Many of the genes we have uncovered in this screen have human homologs for which little or nothing is known. Two paralogous proteins, T04A8.4 and F36F2.1, identified here share considerable homology to the human protein striated muscle activator of Rho signaling (STARS). I demonstrated that these proteins are expressed in body wall muscle and are required for proper actin and myosin localization in muscle. In addition, T04A8.4 appears to form a synthetic genetic interaction with mua-6 and unc-22 and F36F2.1 may form a genetic interaction with cdc-42.

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