We have previously reported a reverse genetic screen to identify genes functioning in germline development (Midwest C. elegans meeting 2000). Briefly, subtracted cDNA probes either enriched for or deprived of the germline-specific transcripts were hybridized to the high-density cDNA grid containing ~7600 non-overlapping EST clones (~40% of the predicted genes in the genome). One hundred and sixty eight candidate clones were then subjected to RNAi. Of these, 21 resulted in embryonic or larval lethality and 15 showed sterility with a variety of different defects in germline development, which included those that caused unfertilized eggs, masculinization of the germline, underproliferative germ nuclei, pachytene arrest, abnormal diakinesis, degeneration of the germ nuclei, and tumorigenesis of the germline (collaboration with M. Mochii, N. Ueno and Y. Kohara). We focused on two of the clones that showed the sterility phenotype. One was
yk445a8, derived from the T05G5.10 gene encoding a homologue of eIF5A. RNAi of this clone caused sterility similar to the
glp-4 mutant; a small gonad with few undifferentiated germ nuclei was observed. The facts that no excess dead cell was observed in the gonad during all larval stages and that germline-tumorigenesis of the
gld-1;
gld-2 double mutant was suppressed in RNAi worms indicated that T05G5.10 functions in germ cell proliferation. C. elegans has another eIF5A homologue, F54C9.1. RNAi of this gene caused a larval arrest phenotype and escapers showed sterility with the abnormal appearance of spermatheca and uterus. We used in situ hybridization and Northern analysis to analyze the germline-specific expression pattern of T05G5.10 and the global expression pattern of F54C9.1. Although eIF5A was originally isolated as a candidate translation initiation factor, recent studies have suggested a role of eIF5A in many aspects of RNA metabolism, including nuclear export, cytoplasmic degradation or translation. We speculate that germline-specific eIF5A is involved in regulation of a set of transcripts essential for the proliferation of germ nuclei. Now we are working to clarify the cellular localization and the functional differences between these two homologues. The other clone in which we are interested is
yk519f1. It corresponds to the F35G12.10 gene, which encodes a homologue of the ATP synthase b subunit. The RNAi of F35G12.10 caused many germ nuclei to arrest at the pachytene stage and spermatocyte-like nuclei were occasionally observed. F35G12.10 shows a strong similarity with another C. elegans gene, F02E8.1. RNAi of F02E8.1 caused larval arrest with complete penetrance, consistent with F02E8.1 functioning as a subunit of ATP synthase, a well-known complex involved in mitochondrial energy generation. Northern analysis of staged worms showed that F35G12.10 was expressed in later larval stages and in adults and F02E8.1 was expressed at all stages. Now we intend to determine if the germline-specific ATP synthase b subunit regulates germline fate through the function of mitochondria.