The
gld-1 gene has multiple functions in germline development.
gld-1 has an essential function in female meiotic prophase progression and oocyte differentiation. In
gld-1 null hermaphrodites, female germ cells enter the meiotic pathway and progress to early pachytene. However, pachytene nuclei inappropriately leave meiotic prophase and proliferate ectopically producing a germline tumor (Francis et al., 1995a, b). GLD-1 contains an RNA binding domain called a KH motif. The ~100 amino acid KH motif resides within an ~200 amino acid region that is highly conserved between GLD-1 and the mouse Quaking and Sam68 proteins. The importance of the GLD-1 KH motif, and presumably it's RNA binding activity, is indicated by missense mutations in the KH motif that disrupt function (Jones and Schedl, 1995). GLD-1 is a cytoplasmic protein (Jones et al., 1996), suggesting that it binds to a subset of cytoplasmic RNAs to regulate their translation and/or stability. In the absence of GLD-1, either in wild-type hermaphrodites during late oogenesis when the protein is eliminated or throughout the germline in
gld-1 null mutants, regulation of RNA translation/stability by GLD-1 is lost leading to altered target RNA activity. Currently, no obvious GLD-1 target RNAs have been identified from genetic studies. We are employing a biochemical and a cell biological approach to identify in vivo RNA targets of GLD-1. For the biochemical approach, we are using immunoprecipitation to purify RNAs that bind to GLD-1. A FLAG-tagged GLD-1 (GLD-1/FLAG) transgene has been used to rescue a
gld-1 null mutant. Cytoplasmic lysates are prepared and GLD-1/FLAG is immunoprecipitated (IP) with either mouse anti-FLAG Ab or mouse IgG and the bound proteins are eluted with FLAG peptide. RNA is then purified from the elutate and converted to cDNA. cDNAs from both the FLAG IP and the control IgG IP are currently being characterized by DNA sequencing. The cell biological approach is based on the hypothesis that GLD-1 is a translational regulator and that in
gld-1 null mutants certain mRNAs become mistranslated. We screened antisera against a number of proteins for gain/loss of staining in
gld-1 null compared to wild-type hermaphrodites. Antisera has been prepared against a peptide that is specific for the C. elegans CDC2 homolog (
ncc-1) which appears to be required, based on RNAi studies, for oocyte meiotic maturation/ initiation of the meiotic divisions (Boxem and van den Heuvel, 1997; Ashcroft et al., 1997). From antibody staining of wild-type hermaphrodite gonads, CDC-2 protein accumulates to high levels in nuclei of growing oocytes in the proximal arm: accumulation is graded with small oocytes containing the least and full grown oocytes containing the most. Interestingly, GLD-1 protein accumulation in wild-type shows a reciprocal pattern: low levels of staining in small oocytes and no staining in full grown oocytes. In the distal gonad of wild type hermaphrodites CDC-2 staining is found at very low levels in early meiotic prophase. By contrast, staining of
gld-1 null hermaphrodites shows premature strong nuclear CDC-2 accumulation in the transition zone, the pachytene region and the ectopically proliferating germ cells. Western blot analysis shows a significant increase in CDC-2 accumulation in
gld-1 null adults compared to wild-type. Thus, CDC-2 levels appear to be regulated by GLD-1. We are currently trying to distinguish whether this regulation is direct or indirect.