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

Abstract

Post-transcriptional regulation is particularly prominent during the maternal-to-zygotic transition (MZT), a developmental phase during which a large proportion of maternally provided mRNAs is repressed and cleared from metazoan embryos. RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory machinery. We show that the ORB2 RBP, the Drosophila ortholog of human Cytoplasmic Polyadenylation Element Binding Protein (hCPEB) 2-4 protein subfamily, binds to hundreds of maternally provided, rare-codon-enriched mRNAs in early embryos; that ORB2 targets are translationally repressed and unstable during the MZT; identify a U-rich motif enriched in ORB2 targets' 3'UTRs; and show that this motif confers ORB2 binding and repression to a luciferase reporter mRNA in S2 tissue culture cells. When tethered to a luciferase reporter, ORB2 and hCPEB2 (but not ORB and hCPEB1) repress translation; the C-terminal Zinc-binding ('ZZ') domain of ORB2 is necessary and 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. ORB2-targets significantly overlap with those previously identified for the repressive RBP, Smaug (SMG). Analysis of the early embryo's translatome in the presence or absence of the endogenous ZZ domain shows that mRNAs bound by ORB2 but not by SMG move onto polysomes upon ZZ domain deletion whereas co-bound transcripts do not, consistent with co-regulation of the latter set of transcripts by both RBPs. Our results assign a function to the ZZ domain and position ORB2 in the post-transcriptional network that regulates maternal transcripts during the Drosophila MZT.

Keywords: Cup; Cytoplasmic Polyadenylation Element Binding Protein (CPEB); ORB2; RNA-binding protein (RBP); S2 cell; embryo; maternal-to-zygotic transition (MZT); post-transcriptional regulation; pre-initiation complex (PIC); translational regulation.

Figure 1.. RIP-seq identifies over 450 ORB2 targets in 0-3 hr embryos.

(a) Domain structure of ORB2B and location of the antigen used to screen for phage-displayed synthetic antibodies. (b) Scatterplot depicting the Log₂(Fold Enrichment compared to Input) for all genes identified in the RIP-seq analysis; each point represents a single gene. Values represent the average enrichment from three independent biological replicates. ORB2 targets (red) are defined as having fold enrichment > 1.5 in the anti-ORB2 RIP-seq compared to input and a ratio of ORB2/Control > 1.5, with an FDR adjusted P-value < 0.05; genes enriched in both ORB2 and control RIP-seq (green) are defined as having fold enrichment > 1.5 in both anti-ORB2 and control RIP-seq, with a ratio of ORB2/Control < 1.5 and an FDR adjusted P-value < 0.05; Control enriched transcripts (blue) are defined as having fold enrichment < −1.5 in the control RIP-seq, fold enrichment < −1.5 in the anti-ORB2 RIP-seq, and an FDR adjusted P-value < 0.05. Dotted lines indicate ±1.5 fold-change thresholds. (c) Violin and boxplots depicting the distribution of transcript abundance as measured by average TPM from three biological replicates based on target status as described in (A). Wilcoxon Rank Sum Test with Bonferroni multiple-test correction: ** = P < 10⁻¹⁰; *** = P < 10⁻¹⁵. (d) Validation of RIP-seq targets by RT-qPCR; bar graphs depict the log₂(fold enrichment) in an anti-ORB2 RIP-RT-qPCR compared to control RIP-RT-qPCR; P-values given are derived from Wilcoxon Signed Rank Test comparing ORB2 RIP-RT-qPCR to control RIP-RT-qPCR.

Figure 2.. ORB2 target transcripts are enriched in translationally repressed, unstable, rare-codon containing transcripts during the MZT.

Violin plots and boxplots depicting the relationship between ORB2-association and translational status, change in poly(A) tail length, and change in transcript abundance. Non-target genes include all expressed genes that were not defined as ORB2 targets in the RIP-seq analysis. (a) Translation efficiency, calculated from ribosome footprinting performed on 0-1 hour embryo extracts (Eichhorn et al. 2016). (b) Translation efficiency, calculated from ribosome footprinting performed on 2-3 hour embryo extracts (Eichhorn et al. 2016). (c) Translation efficiency as measured by ribosome profiling performed on 0-2 hour embryo extracts (Dunn et al. 2013). (d) Translation index, calculated from polysome gradients performed on 0-3 hour embryo extracts (Chen et al. 2014). (e) Change in transcript poly(A) tail length, calculated by comparing 0-1 hour and 2-3 hour embryo extracts, as measured by PAL-Seq (Eichhorn et al. 2016). (f) Change in abundance, calculated by comparing 0-1 hour and 2-3 hour embryo extracts, as measured by RNA-Seq (Eichhorn et al. 2016). (g) MELP score, calculated for all annotated CDS longer than 80 codons on FlyBase 6.58 by coRdon with ribosomal proteins as the references for codon optimization, and the isoform with minimum score (the worst expressivity) is chosen to represent each expressed gene. (h) CAI score, calculated for all annotated CDS longer than 80 codons on FlyBase 6.58 by local version of CAIcal with the Drosophila melanogaster codon usage table from the Kazusa codon usage database, and the isoform with minimum score (the most enriched for rare codons) is chosen to represent each expressed gene. Wilcoxon Rank Sum Test: ** = P < 10⁻¹⁰; *** = P < 10⁻¹⁵.

Figure 3.. ORB2 binding motifs predicted from RIP-seq confer translational repression on a reporter mRNA in S2 cells.

(a) The #ATS de novo motif discovery program (Li et al. 2010) was performed on the results from the anti-ORB2 RIP-seq in this study. #ATS identified NUUGUWU as the ORB2 recognition element (ORE) enriched in ORB2 targets. (b) The transcriptome was evaluated for accessible enrichment of the NUUGUWU motif and each transcript was scored based on the number and accessibility of these motif sites. ORB2 targets had significantly higher scores compared to non-targets, as assessed by Wilcoxon rank sum test. *** = P < 10⁻¹⁵ (c) Diagram depicting the reporter firefly luciferase mRNA with six copies of different ORB2-binding motifs in the 3’UTR. (d) Bar graph depicting the enrichment of the luciferase reporter mRNA in an anti-ORB2 RIP-qPCR compared to Renilla luciferase mRNA, normalized to a reporter without ORE motifs. Mutation of the OREs or deletion of the RNA-binding domain (RRM) abrogate binding to the mRNA. (e) Bar graph depicting the ratio of luminescence between firefly luciferase and Renilla luciferase. Mutation of the OREs or deletion of the RNA-binding domain (RRM) abrogate repression. One-way Kruskal-Wallis non-parametric test with Dunn multiple test correction: * = P < 0.05.

Figure 4.. The ORB2 and hCPEB2 ZZ domain but not the ORB and hCPEB1 ZZ domain repress a luciferase reporter.

(a) Diagram depicting the tethering assay in which a polypeptide of interest fused to the BIV-Tat domain is tethered to a firefly luciferase reporter mRNA carrying six tandem repeat TAR elements in the 3’UTR. (b) Bar graph depicting the expression of the reporter firefly luciferase (normalized to co-transfected Renilla luciferase and normalized to steady state RNA levels) when tethering whole ORB2 or ORB2 fragments compared to GFP control. (c) Bar graph depicting the expression of the reporter firefly luciferase mRNA measured by RT-qPCR (normalized to co-transfected Renilla luciferase mRNA) when tethering whole ORB2 or ORB2 fragments compared to GFP control. Reporter mRNA levels are unaffected by ORB2 ZZ or ORB2ΔZZ. (d) amino acid sequence alignment of the ZZ domains of ORB2, ORB, and their human orthologs, hCPEB1 and hCPEB2. The arrowhead denotes the junction between the RBD and ZZ domains. (e) Bar graph depicting the expression of the reporter firefly luciferase (normalized to co-transfected Renilla luciferase) when tethering different CPEB ZZ domains. Only ORB2 and hCPEB2 repress. (f) Bar graph depicting the expression of the reporter firefly luciferase when ORB ZZ chimeric domains harboring substitutions from the ORB2 ZZ domain, are tethered. One-way Kruskal-Wallis non-parametric test with Dunn multiple test correction: * = P < 0.05.

Figure 5.. Anti-ORB2 IP-MS from early embryo extracts identifies known translation repressors and the PIC as binding partners.

(a-b) Scatterplots depicting all proteins identified in the IP-MS performed in the presence of RNase. Enrichment is expressed as the Log₂ ratio of the average spectral counts detected in the ORB2 IP compared to the average spectral counts detected in the control IP. Protein interactors were identified by SAINT (with a SAINT score ≥ 0.95 and BFDR < 0.01), shown in red. (a) IP-MS performed on embryos laid by w¹¹¹⁸ females, (b) IP-MS performed on embryos laid by orb2^ΔZZ females. (c) Interaction network depicting the connections between components of the Cup repressive complex and other translational repressors identified as ORB2 interactors by SAINT analysis. (d) Interaction network depicting the connections between components of eukaryotic PIC identified as ORB2 interactors by SAINT analysis. Interaction networks were produced by the GeneMANIA app for Cytoscape (Franz et al. 2018); size of node indicates average number of spectral counts obtained in the ORB2 IP-MS; thickness of edges indicates strength of interaction assigned by GeneMANIA.

Figure 6.. Deletion of the ORB2 ZZ domain results in derepression of target RNAs in the early embryo.

(a) Violin and boxplots comparing target and non-target abundance as determined by RNA-Seq in 0-1, 1-2 and 2-3 hour old embryos laid by w¹¹¹⁸ and orb2^ΔZZ females. No significant difference was detected for either genotype at any of the time-points. (b) Violin plots and boxplots comparing target and non-target translation index as determined by polysome gradients followed by RNA-Seq in 0-1, 1-2 and 2-3 hour old embryos laid by w¹¹¹⁸ and orb2^ΔZZ females. ORB2 targets were significantly derepressed in 0-1 hour old orb2^ΔZZ embryos relative to w¹¹¹⁸. Log2(Ratio of mean normalized counts of pool 3/mean normalized counts of pool 1) was calculated for each transcript. Wilcoxon Rank Sum Test: ** = P < 0.01.

Figure 7.. ORB2 targets which are co-bound by SMG are not repressed in the early embryo.

(a-d) Violin and boxplots comparing the polysome association of ORB2 and SMG co-targets (a), ORB2 targets (b), SMG targets (c), and non-targets (d), as determined by polysome gradients followed by RNA-Seq in 0-1 hour old embryos laid by w¹¹¹⁸ control (WT) and orb2^ΔZZ females. Wilcoxon Rank Sum Test ** = P < 0.01.