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: For each replicate of RIP-seq, 1mL of 0-3h embryo extract (prepared as described above) was supplemented with Triton X-100 to 0.1% final concentration, then diluted in an equal volume of lysis buffer with 0.1% Triton X-100 and cleared again by centrifugation at 4°C, 13 000 RPM for 15 minutes. The supernatant was incubated for 3 to 4 hours at 4°C with end-over-end rotation with 100µL of anti-FLAG M2 affinity gel beads (Sigma) which had been pre-incubated at 4°C with 50µg of purified anti-ORB2 E8 synthetic antibodies blocked with 5µg/µL BSA. After incubation, the lysates were decanted and beads were washed four times with lysis buffer supplemented with 0.1% Triton X-100 and then three times with equilibration buffer: 100 mM NaCl, 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2. Fabs and associated RNPs were eluted from the beads with 200 µL of 200µg/µL FLAG-peptide (Sigma) suspended in equilibration buffer at 4°C for 20 minutes. RNA was isolated from the eluate using TRI Reagent (Sigma) according to the manufacturer's instructions. RNA was prepared for sequencing using Ribo-Zero Gold rRNA depletion kit and TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to manufacturer's instructions. Libraries were sequenced using the HiSeq 2500 System, V4 Chemistry (Illumina), with paired-end reads 125nt in length.