Drosophila early embryos LC-MSMS

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts.The conserved RNA-binding protein family CPEB has been shown to mediate canonical cytoplasmic polyadenylation of target transcripts. We have previously reported evidence for RNA-interference factor Dicer-2 as a component of a non-canonical complex, that operates independent of CPEB in Drosophila. In this study, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP‐Seq analysis we identify hundreds of potential Dicer-2 target transcripts, ~60% 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 and mass spectrometry revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA‐independent complex with Dicer‐2, and mediate interactions of Dicer‐2 with Wispy. Functional poly(A)‐test analyses showed that Twenty‐four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer‐2 targets. Our results reveal components of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.