Germline and early embryonic development of higher eukaryotes rely extensively on post-transcriptional mRNA regulation. The control mechanisms of translational mRNA activation or repression are crucial for cell fate specification and seem to be largely conserved. For example,
gld-3 (germline development defective-3) of C. elegans, a member of the conserved Bicaudal-C family of RNA-binding proteins, is an important regulator of germline and embryonic development (1). GLD-3 is part of larger mRNP complexes, which are expected to regulate specific sets of target mRNAs. Interestingly, two distinct cytoplasmic poly(A) polymerases (PAPs), GLD-2 and GLD-4, are proposed to rely on GLD-3 activity (2). In our model, GLD-3 provides an RNA-binding activity to both non-canonical PAPs and may even serve as a platform for other regulatory proteins to bind and influence the target specificity of different GLD-3-containing complexes. However, it is unclear what the individual complexes are composed of and what the respective target mRNAs are. To test directly the RNA-binding ability of GLD-3 we employed RNA homopolymer binding assays. GLD-3 specifically bound to poly(G) RNA and not to poly(U), (C) or (A), a feature shared with Drosophila Bicaudal-C (3). To identify in vivo mRNA targets of the GLD-2/3 complex we used a candidate gene approach and performed RNA co-immunoprecipitation (RNA co-IP) experiments. We identified and characterized
gld-1 as the first mRNA target of the GLD-2/GLD-3 PAP in collaboration with the Kimble lab (4). However, we learned from this study that
gld-1 mRNA translation depends only partially on GLD-2 PAP activity and that
gld-1 mRNA regulation is fully compensated for in
gld-3 mutants. Recently, we found that
gld-1 mRNA is a target of a second cytoplasmic PAP, the GLD-4/GLS-1 complex (2). We now report that GLD-3 is also a member of the GLD-4/GLS-1 PAP in vivo, suggesting that
gld-1 mRNA activation shares a common component, GLD-3, and two in parallel acting but distinct PAP enzymes, GLD-2 and GLD-4. Finally, to find more GLD-3-RNP targets in an unbiased way we performed RIP-CHIP (RNA co-IP and microarray analysis) experiments. We have now a preliminary list of ~70 potential GLD-3 targets, which are currently validated with various biochemical techniques. The confirmation of their biological significance to be a target of GLD-3 and its associated PAPs in vivo is still pending. (1) Eckmann CR et al. (2004) Genetics 168(1),
pp147-60. (2) Schmid M et al. (2009). Genes and Dev. (in press). (3) Saffman EE et al. (1998) Mol Cell Biol. 18(8),
pp4855-62. (4) Suh N et al. (2006) Proc Natl Acad Sci USA 103 (41),
pp15108.