GLH-1 and GLH-2 represent two germline-specific, putative RNA helicases that are components of the P granules at all stages of the worm life cycle.
glh-1 RNA and protein are found in both the male and hermaphrodite germlines, while
glh-2 RNA and protein are predominately associated with only the hermaphrodite germline. With the use of confocal microscopy, affinity-purified antibodies specific to GLH-1 and GLH-2 show that these proteins co-localize; they also co-localize with previously described anti-P granule antibodies. Antisense injections of either
glh-1 or
glh-2 RNAs result in ~10% of the injected progeny becoming sterile adults with disorganized gonads that lack anti-P granule antibody staining. The antisense results suggest a critical role for the GLHs in the germline. Attempts to isolate
glh-1 and
glh-2 mutants by reverse genetics are in progress. Two additional potential germline-specific RNA helicase genes,
glh-3 and
glh-4, have recently been identified, based on sequence similarities to
glh-1 and
glh-2 and the fact that all four potential glhs map on the left side of LGI. The four GLHs possess the eight motifs common to ATP-dependent RNA helicases. We are performing in situ hybridization and antibody studies to determine if both
glh-3 and
glh-4 are germline- specifiC. Preliminary in situ hybridization studies indicate
glh-3 is germline-specific, with immuno-cytochemistry tentatively identifying GLH-3 as an additional component of the P granules. Experiments to phenocopy
glh-3 and
glh-4 are currently being performed. The four GLHs, each containing retroviral-like zinc fingers, are unique when compared to germline-specific helicases reported in other organisms. The zinc fingers may enable the GLH proteins to bind specific RNAs. Each GLH has a different number of these short CCHC fingers. In addition, imperfect glycine-rich repeats dominate the amino half of GLH-1, -2, and -4. Biochemical analyses for helicase and RNA binding activities are ongoing. Our working model is that the GLHs sequester and prevent the degradation of germline-specific RNAs in the developing embryo. The GLHs may also facilitate the translation of these RNAs. Discerning the role of the GLH P granule components in germline determination and development should form a basis for understanding P granule function in the C. elegans germline.