The C. Elegans GLHs and P Granule Interactions

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Bennett 0078169 The germline is responsible for carrying on the species; without a germline, sexually-reproducing organisms would not exist. In most animals germline precursor cells are set aside during embryogenesis, with the precursors dividing later in life. After proliferation, these cells undergo meiosis to produce male and female gametes, the eggs and sperm of the adult. In the model organism, the free-living soil nematode Caenorhabditis elegans, the germline is established very early in development. By the fourth cell division of the embryo, a single cell, called P4, is the founder cell for the entire germline. Throughout the life of the worm, cytoplasmic complexes of RNA and protein, called P granules, are exclusive to the germline lineage. Similar germ granules are found in the germline precursor cells of all higher organisms that possess a germline. The Bennett laboratory has identified four germline RNA helicases, the GLHs, a family of putative ATP-dependent enzymes that are exclusive to P granules throughout the life of the worm. Similar germline RNA helicases are found in the germ granules of flies, frogs, mice and men. This application proposes to determine the genetic interactions between the four glh genes, as well as identifying the RNAs and proteins that interact with these RNA helicases in P granules. Based in part on multiple CCHC zinc fingers that are unique to the GLHs, it is hypothesized that each GLH may associate with a particular set of RNAs and proteins. The work to be carried out will include the isolation of a null mutation in the glh-4 gene, the only glh gene for which a loss-of-function mutation remains to be isolated. Relevant multiple glh mutant strains will be generated, including the glh-1 glh-4 double mutant which RNAi (RNA interference) studies indicate will result in complete sterility. RNAs bound by the GLHs will be enriched for with the SELEX technique, while proteins that interact with the GLHs will be identified in yeast two-hybrid assays. The yeast two hybrid screens have already identified a putative MAP kinase that appears to interact with GLH-3, as well as several other candidate proteins. These analyses will reveal how the GLHs, with their RNA and protein partners, affect germline determination, as well as the establishment and maintenance of the species. This work in worms is expected to relate to reproductive strategies in all organisms, including humans.