Article Figures & Data
Figures
- Figure 1. Identification of Dicer-2 mRNA targets.
(A) Representative image of Dicer-2 immunoprecipitation. IgG, negative control. (B) Volcano plots showing RNA enrichment in Dicer-2 IPs with respect to input (left) or IgG (right). Red, fold enrichment > 2; orange, fold enrichment between 1.5 and 2; gray, not enriched. Toll (Tl) and bicoid (bcd) mRNAs are indicated. (C) Overlap between the two lists of potential Dicer-2 targets. (D) RNA types bound by Dicer-2. (E) GC-content of Dicer-2 targets (red) compared with non-targets (black). (F) Overlap of Dcr-2 and Wispy mRNA targets reported in three studies. Potential Dicer-2 polyadenylation targets are highlighted in bold. (G) Validation of RIP-seq by RT-qPCR. Selected targets of the Dcr-2/Wispy overlap were chosen. The enrichment was normalized to sop (RpS2), a ribosomal protein mRNA which does not undergo cytoplasmic polyadenylation. Toll and bicoid mRNAs (green) were used as positive controls, whereas RpL32 and RpS3 mRNAs (gray) were used as non-targets. Bar plots represent the average of three biological replicates. Error bars represent SD. Significance was assessed by t test (*P < 0.05, **P < 0.01).
Source data are available for this figure.
- Figure 2. Identification of the Dicer-2 protein interactome.
(A) Schematic representation of the strategy to obtain Dicer-2 null embryos. (B, C) Comparison of wild-type (w1118, wt) and Dicer-2 null (fsX) embryos (B) or αDicer-2 versus IgG IPs (C). In both cases, an immunoblot analysis of three replicates after treatment or not with RNase I is shown on the left, and the overlap between SAINT and Top3 analyses is shown on the right. (D) The Dicer-2 high confidence interactome, defined as the overlap between proteins identified in (B) and (C) (86 proteins). (E) Gene Ontology analysis of the Dicer-2 high-confidence interactome using Flyenrichr under the term “Biological Process.”
Source data are available for this figure.
- Figure 3. Dicer-2 interacts with Wispy through Ataxin-2 and Twenty-four.
(A) Comparison between the Dicer-2 high-confidence interactome and the Drosophila RBPomes identified in 0–2 h embryos (Wessels et al, 2016) and across the maternal-to-zygotic transition (Sysoev et al, 2016). (B) Validation of Dicer-2 partners by co-immunoprecipitation. Dicer-2, Tyf, Atx2, and Wispy interact in an RNA-independent fashion. UNR was used as a negative control. (C) Schematic representation of the crosses performed to obtain Atx2- and Tyf-depleted extracts (top) and efficiency of depletion (bottom). (D) Interaction of Dicer-2 with Wispy is mediated by Atx2 and Tyf. Dicer-2 was immunoprecipitated from wild-type, Atx2-, and Tyf-depleted extracts in the presence of RNases. Co-IP of the indicated factors was assessed by Western-blot. IgG IP using wild-type extracts served as background control. UNR was used as loading control. The asterisk denotes residual signal from Wispy detection. (E) Confirmation of complex formation and co-dependencies by Ataxin-2 pull-down. (D) Immunoprecipitation was performed as in (D). Bicaudal-C was used as loading control. (F) Model of the complex.
Source data are available for this figure.
- Figure 4. Ataxin-2 and Twenty-four function in cytoplasmic polyadenylation of a subset of Dicer-2 targets.
(A) Efficiency of polyadenylation of Dicer-2 targets in wild-type (w1118) versus Dicer-2 null, Atx2-depleted, or Tyf-depleted embryos. The average of at least three independent experiments is shown on the right. Error bars represent SD. Significance was assessed using one-way ANOVA test with Bonferroni correction (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Model for the embryonic noncanonical cytoplasmic polyadenylation complex. Interactions of Dicer-2 with Wispy, Atx2, and Tyf are depicted. Atx2 has been previously shown to interact with PABP.
Source data are available for this figure.
Supplementary Materials
Article Figures & Data
Figures
Source Data for Figure 1[LSA-2022-01417_SdataF1.1.pdf][LSA-2022-01417_SdataF1.2.xlsx]
Source Data for Figure 2[LSA-2022-01417_SdataF2.pdf]
Source Data for Figure 3[LSA-2022-01417_SdataF3.pdf]
Source Data for Figure 3[LSA-2022-01417_SdataF4.1.pdf][LSA-2022-01417_SdataF4.2.xlsx]
Supplementary Materials
Table S1. Dcr-2 mRNA targets. [LSA-2022-01417_TableS1.xlsx]
Table S2. Dcr-2 protein interactome. [LSA-2022-01417_TableS2.xlsx]
