Early development is governed in part by a post-transcriptional regulatory network that acts through the 3'' untranslated regions (3'' UTR) of targeted transcripts. This network requires RNA-binding proteins with weak sequence specificity, confounding attempts to identify regulatory targets. To date, few direct regulatory targets of these proteins have been identified.
glp-1, which encodes a Notch-like receptor critical for development, is directly regulated by at least two RNA-binding proteins, POS-1 (1) and GLD-1 (2). Both are co-expressed in the posterior of the early embryo, while
glp-1 mRNA is present throughout the early embryo. Embryos lacking either protein exhibit ectopic GLP-1 expression (1,2). Previously, we determined the sequence specificity of both POS-1 (3) and GLD-1 (4) and predicted binding sites for each protein in 3'' UTRs. The region of the
glp-1 3'' UTR required for translational repression in embryos contains a GLD-1 binding site flanked by adjacent POS-1 binding sites. The close proximity of these sites and the similar GLP-1 expression patterns in embryos lacking POS-1 or GLD-1 suggest that both proteins co-repress
glp-1 translation. Using in vitro methods, we show that POS-1 does not cooperate with GLD-1, but instead antagonizes GLD-1 binding to the
glp-1 3'' UTR. In addition, we show that POS-1 is homodimeric, suggesting that specific recognition of
glp-1 mRNA by POS-1 is driven by self-association. Other mRNA-protein complexes may also play a role in regulating
glp-1, as the
glp-1 3'' UTR contains two MEX-3 binding sites that surround the POS-1 and GLD-1 sites. This cluster of sites is conserved across nematode species, and is essential for proper
glp-1 expression (2). Thus, we hypothesize that conserved clusters of binding sites provide the basis for specific mRNA recognition. To identify other clusters of conserved binding sites, we have built a genome-wide database of predicted RNA-binding protein sites, 3'' UTRs, and conservation scores. Well-conserved binding sites tend to cluster in 3'' UTRs and may be more predictive of regulatory elements than individual binding sites alone. 1. Ogura K, et al. Development 2003:2495-503 2. Marin VA, Evans TC. Development 2003:2623-32 3. Farley BM, et al. RNA 2008:2685-97 4. Ryder SP, et al. Nat Struct Mol Bio 2004:20-8.