Ataxin-2, Twenty-four and Dicer-2 are components of a non-canonical cytoplasmic polyadenylation complex

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative non-canonical, CPEB-independent complex in Drosophila, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP-Seq analysis we identify hundreds of novel Dicer-2 target transcripts, ~50% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Functional analysis indicate that both factors form an RNA-independent complex with Dicer-2, and are required for cytoplasmic polyadenylation of Dicer-2 targets. Our results reveal the composition of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis. Overall design: RNA immunoprecipitation-sequencing (RIP-Seq) was performed to indentify Dicer-2 RNA targets in Drosophila melanogaster early embryo extracts. Three types of samples, in sextuplicates, were produced: (1) RNA eluate from specific immunoprecipitation against Dicer-2, either compared to (2) RNA from non-specific IgG immunoprecipitation (control) or (3) RNA from Input samples.