It is truly amazing that you and I were once each a fertilized egg no bigger than the period at the end of this sentence. Much of the basic knowledge of how humans develop has come from research using simple organisms that allow molecular and genetic analyses. The Bennett laboratory is interested in those genes critical to the establishment and maintenance of the germline. In most organisms germline precursor stem cells are set aside during embryogenesis; these precursors will proliferate later in development and some of the daughters will undergo meiosis to produce male and female gametes. In the free-living soil nematode, Caenorhabditis elegans, the germline is established very early in development. By the fourth cell division a single cell, P4, has become the germline founder cell. During these first divisions and throughout the life of the worm, non-membrane-bound cytoplasmic complexes called P granules are specifically present in the germline lineage. Similar germ granules are found in all higher eucaryotes that have a dedicated germline. This laboratory was one of the first to identify P granule components, reporting that the GLH proteins, germline RNA helicases, localize to P granules. The GLHs are critical to the development of the germline lineage.
The long-term goal of this work is to determine the biochemical function of the GLH proteins. With previous NSF funding, mutant strains for each of the four glh genes have been generated. While neither glh-2 nor glh-3 seems to have a role in germline development under laboratory conditions, the combined loss of glh-1 and glh-4 by RNAi (RNA interference) causes a dramatic block in oogenesis at meiosis and results in complete sterility. With renewed funding the genetic analysis of each glh mutant and the construction of a glh-1;glh-4 double mutant strain will be carried out, Specific Aim 1. Upstream regulators of the GLH proteins are the focus of Specific Aim 2. A yeast two-hybrid screen previously identified proteins that interact with the GLHs including: KGB-1, a novel serine/threonine kinase; CSN-5, a CSN signalosome component; and PAN-1, a novel protein containing F-box signatures. In other organisms proteins similar to each of these GLH interactors are implicated in protein degradation pathways; therefore, it was predicted these partners regulate GLH protein levels. Recently the sterile kgb-1(um3) allele, which is likely a null, has been used to detect a genetic interaction between GLH-1 and KGB-1. When KGB-1 is missing, GLH-1 levels are inappropriately high. Suppression of kgb-1 sterility by csn-5 RNAi has also been observed, indicating these two GLH partners interact with each other. These and several other findings led to the hypothesis that KGB-1 targets GLH proteins for degradation and that the CSN complex normally protects GLH protein stability. In Specific Aim 2 a model of how GLH protein stability is regulated will be tested.
The intellectual merit of this application is based upon using the model organism Caenorhabditis elegans for lineage determination studies, arguably the most appropriate model to address this area of inquiry. Germline RNA helicases are the most conserved components of vertebrate and invertebrate germ granules, adding to the likely importance of the GLH proteins in evolution. Since most other C. elegans P granule components are novel, the GLH germline helicases should provide a key link from P granules in worms to the germ granules of flies, frogs and humans. The broad impacts of this project are two-fold. This work in C. elegans relates to the important topics of reproduction, fertility and the maintenance of the species. Because nematodes are major contributors to agricultural and world health problems, with Ascaris being the most common helminthic parasite in the world, this work has the potential to be translated into benefits for mankind (4). The project will also provide training to a postdoctoral fellow, graduate and undergraduate students. With the previous NSF award, three underserved minority undergraduates worked in the Bennett laboratory. Publications from the last award resulted in four undergraduate authors. It is anticipated the trainees supported by this award will have similar success from their research experiences.
