The ORB2 RNA-binding protein negatively regulates its target transcripts during the Drosophila maternal-to-zygotic transition via its functionally conserved Zinc-binding 'ZZ' domain

RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory machinery. We show that the ORB2 RBP, the Drosophila ortholog of vertebrate Cytoplasmic Polyadenylation Element Binding Protein 2 (CPEB2), binds to hundreds of maternally provided mRNAs in early embryos, identify a U-rich motif enriched in ORB2's targets, and show that this motif confers ORB2 binding and repression to a luciferase reporter mRNA in S2 tissue culture cells. ORB2's target transcripts are translationally repressed and unstable during the maternal-to-zygotic transition (MZT), a developmental phase during which a large proportion of maternally provided mRNAs are repressed and cleared. We show that, when tethered to a luciferase reporter, ORB2 and its human homolog CPEB2 (but not ORB and CPEB1) repress translation and that the C-terminal Zinc-binding ('ZZ') domain of ORB2 is sufficient for repression. ORB2 interacts with a suite of post-transcriptional regulators in early embryos; a subset of these interactions is lost upon deletion of the ZZ domain, notably with the Cup repressive complex. Analysis of the early embryo's translatome in the presence or absence of the endogenous ZZ domain, shows that ORB2's targets move onto polysomes upon ZZ domain deletion, indicating that this domain mediates translational repression of ORB2's targets during the MZT. Together, our results assign a function to the ZZ domain and support a significant role for ORB2 in post-transcriptional regulation of maternal mRNAs during the Drosophila MZT. Overall design: Total RNA-seq profiling of embryo of wild type and orb2-delta-zz D. melanogaster at 0-1hr, 1-2hr and 2-3hr of post egg-lay.