The Novel Cytoplasmic Poly(A) Polymerase GLD-4 Regulates Germ Cell Fate Decisions in C. elegans. Mark Schmid and Christian Eckmann. The adult C. elegans germ line is an excellent model to study the fine balance existing between proliferation and differentiation, forces necessary for tissue generation and homeostasis. A constant mitotic germ cell pool is maintained through proliferative signals emanating from the distal tip cell, which functions as a somatic stem cell niche. Further proximally, germ cells enter meiosis and subsequently differentiate into gametes in a sex-specific manner. We are interested in the molecular controls of these germ cell fate decisions. Our work focuses on post-transcriptional RNA mechanisms. It is the action of conserved translational repressors and activators that build a molecular switch governing the entry from mitosis into meiosis. One of the important players is GLD-3, a Bicaudal-C type RNA-binding protein, which acts in a complex together with GLD-2, a cytoplasmic poly(A) polymerase, to promote entry into meiosis. Germ cells in
gld-2 or
gld-3 loss-of-function mutants over proliferate and fail to enter meiosis appropriately. Further analysis of
gld-3 led us to discover
gls-1, which encodes a protein that interacts with GLD-3 and appears to have opposite functions from the
gld-3 gene. Germ cells in
gls-1 mutants exit mitosis and enter meiosis precociously.. In order to understand the molecular role of this antagonism we employed a yeast 2-hybrid screen using GLS-1 as bait and recovered multiple cDNAs encoding a novel conserved protein, termed GLD-4. Database searches revealed a strong similarity of GLD-4 to members of the cytoplasmic poly(A) polymerase protein family. GLD-4 contains a nucleotidyltransferase domain and a PAP associated domain similar to GLD-2. Also like GLD-2, GLD-4 lacks an obvious RNA-binding domain. We have raised a polyclonal antibody against GLD-4 and find it highly enriched in the germ line where it localizes to the cytoplasm. In particular, we find GLD-4 to be an integral part of P-granules at all stages of embryonic and postembryonic germ cell development, thus suggesting a possible role for GLD-4 in germ cell development.. We carried out RNAi experiments to characterize in vivo roles of
gld-4 and discovered its requirement for germ cell proliferation, a function that seems to be shared with
gls-1. We identified a germ cell proliferation defective mutation in
gls-1(
ef4) which is unable to bind to GLD-4 in Y2-Hybrid experiments but is still expressed. Taken together, we propose that GLD-4 is a putative cytoplasmic poly(A)polymerase that activates mRNAs encoding germ cell fate determinants required for the mitosis/meiosis decision in conjunction with GLS-1 to promote mitosis.