The GLH proteins, Caenorhabditis elegans P granule components, associate with CSN-5 and KGB-1, proteins necessary for fertility, and with ZYX-1, a predicted cytoskeletal protein.

The GLH proteins belong to a family of four germline RNA helicases in Caenorhabditis elegans. These putative ATP-dependent enzymes localize to the P granules, which are nonmembranous complexes of protein and RNA exclusively found in the cytoplasm of all C. elegans germ cells and germ cell precursors. To determine what proteins the GLHs bind, C. elegans cDNA libraries were screened by the yeast two-hybrid method, using GLHs as bait. Three interacting proteins, CSN-5, KGB-1, and ZYX-1, were identified and further characterized. GST pull-down assays independently established that these proteins bind GLHs. CSN-5 is closely related to the subunit 5 protein of COP9 signalosomes, conserved multiprotein complexes of plants and animals. RNA interference (RNAi) with csn-5 results in sterile worms with small gonads and no oocytes, a defect essentially identical to that produced by RNAi with a combination of glh-1 and glh-4. KGB-1 is a putative JNK MAP kinase that GLHs bind. A kgb-1 deletion strain has a temperature-sensitive, sterile phenotype characterized by the absence of mature oocytes and the presence of trapped, immature oocytes that have undergone endoreplication. ZYX-1 is a LIM domain protein most like vertebrate Zyxin, a cytoskeletal adaptor protein. In C. elegans, while zyx-1 appears to be a single copy gene, neither RNAi depletion nor a zyx-1 deletion strain results in an obvious phenotype. These three conserved proteins are the first members in each of their families reported to associate with germline helicases. Similar to the loss of GLH-1 and GLH-4, loss of either CSN-5 or KGB-1 causes oogenesis to cease, but does not affect the initial assembly of P granules.

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans.

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.