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. 2016 Jul 5;7:12128.
doi: 10.1038/ncomms12128.

Global Changes of the RNA-bound Proteome During the Maternal-To-Zygotic Transition in Drosophila

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Free PMC article

Global Changes of the RNA-bound Proteome During the Maternal-To-Zygotic Transition in Drosophila

Vasiliy O Sysoev et al. Nat Commun. .
Free PMC article

Abstract

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.

Figures

Figure 1
Figure 1. Identification of the Drosophila RNA interactome.
(a) Direct RNA binders are CL to mRNAs in living Drosophila embryos, which are subsequently lysed under denaturing conditions. mRNA-protein complexes are purified by hybridization with oligo(dT) magnetic beads and a series of stringent washes. Proteins are released by RNase treatment and are ready for MS analysis. (b) Analysis of total and oligo(dT) by Bioanalyzer 2100 captured RNA shows depletion of abundant ncRNAs. (c) Multifold enrichment of polyadenylated gapdh and ts mRNAs in oligo(dT) bound fractions confirmed by RT-qPCR. On the x axis are indicated the samples in which the amounts of the different RNAs were measured: Input noCL, input CL, eluate noCL, eluate CL. The y axis represents the fold enrichment of RNA amounts in the different samples. RNA amounts in noCL input were defined as 1. Error bars: s.d. See Supplementary Table 1 for information on oligonucleotides used for the analysis. (d) Protein profiles of total embryo and RNA-bound fractions. (e) Analysis of total embryo lysates and oligo(dT) bound fractions by western blotting with antibodies against Vasa, eIF4E, Pabp2, PABPC, Hrp48, tubulin, Y14 and H3. Lysis at 60 °C and 12.5 mM DTT ensures that tubulin background is removed. See Supplementary Table 2 for antibody information. (f) Replicated samples prepared from pre- (0–1 h) and post-MZT (4.5–5.5 h) embryos are mixed to generate three aggregate CL and noCL (control) samples. Proteins are partially digested, TMT labelled and quantified by MS. (g) Scatter plot showing protein abundance ratios CL/noCL in two replicates. Red dots represent proteins significantly enriched in CL samples. (h) Venn diagram comparing numbers of detected and significantly enriched proteins.
Figure 2
Figure 2. Composition of the Drosophila mRNA interactome and its links to development.
(a) Blue bars: numbers of proteins annotated with GO term ‘RNA binding' and other RNA-related GO terms. Green bars: numbers of proteins containing and not containing domains annotated as RNA-binding in the Pfam database. Dark blue: GO term ‘RNA binding'; medium blue: RNA-related GO terms; light blue: GO annotation unrelated to RNA; dark green: known RBD; light green: no known RBD. (b) Balloon plot related to a. (c) Distribution of disordered regions, low complexity domains and repetitive sequences in the total proteome (red), RNA interactome (blue), previously known RBPs (green) and newly discovered RBPs (purple) in the RNA interactome. Two-sample Kolmogorov–Smirnov test P values are listed in the Supplementary Note 3. (d) Five most frequent repetitive sequence patterns in the RNA interactome. (e) Venn diagram showing numbers of proteins belonging to the human (yellow), mouse (pink) and fly (green) interactomes. Of 266 fly-specific RBPs 65 do not have a mammalian ortholog. (f) Blue bars: fly-specific RBPs and RBPs shared with one or more eukaryotic interactomes are compared to RNA-related GO terms (blue bars). Green bars: numbers of proteins with Pfam-annotated known RBDs. (g) Fractions of proteins with embryonic, lethal and sterile phenotypes in the RNA interactome (blue) and the proteome (red). Fischer test P values resulting from comparison of the interactome to the proteome: lethal—0.00346; embryonic—0.03483; sterile—0.0001754.
Figure 3
Figure 3. Validation of CycB and EB1 as RNA-binding proteins.
(a) Optimization of GFP immunoprecipitation. Protein profiles of total embryo lysates, IPs of free GFP and unbound fractions are visualized on a silver stained polyacrylamide gel. Lanes 1–3: serial dilution of total embryo lysate expressing free GFP; lanes 4–6: serial dilution of unbound fractions after GFP-IP; lanes 7–10: GFP immunoprecipitates washed with buffers containing increasing concentrations (none; 0.01%; 0.05%; 0.1%) of SDS. (bd) Lysates containing free GFP, EB1-GFP and GFP-CycB were treated with various concentrations of RNase A. GFP proteins were IPed from noCL and CL embryos, radioactively labelled, separated on polyacrylamide gels and blotted on PVDF membranes. Upper panels: autoradiography of membranes containing IPs of GFP (b), EB1-GFP (c) and GFP-CycB (d) labelled with γ-[32P]-ATP by T4 polynucleotide kinase (see Methods section, PNK assay). Lower panels: visualization of GFP proteins by WB with an anti-GFP antibody.
Figure 4
Figure 4. Study of RNA interactome dynamics during the maternal-to-zygotic transition.
(a) Schematic representation of the MZT. Shaded areas show the time frames considered in our study. (b) Proteins captured from three biological replicates of CL early (0–1 h) and late (4.5–5.5 h) embryos were partially digested, labelled by six different TMT labels and analyzed by MS. (c) Protein profiles of total lysates and RNA-bound fractions recovered from early and late embryos, noCL and CL. (d,e) Ratios of protein abundance late/early in two of the three biological replicates. (d) RNA-bound fractions, (e) total lysates. Red dots: proteins whose abundance significantly changes at FDR 1%; yellow dots: proteins whose abundance changes at FDR 10%. Supplementary Fig. 3a,c includes the same plots, with P values and Pearson correlation values indicated. (f) Scatter plot showing change of average protein abundance early/late in RNA-bound fractions (ordinate) and total protein lysates (abscissa). Grey dots: proteins whose abundance does not significantly change neither in the RNA-bound fraction, nor in the total embryo lysate. Black dots: proteins whose abundance change in the RNA-bound fractions follows the change in the total embryo lysate. Yellow dots: proteins whose abundance in the RNA-bound fraction significantly changes, and is inconsistent with their abundance change in the total protein lysate, suggesting active regulation of their RNA-binding capacity (‘dynamic binders'). (g) Numbers of proteins in each of the dynamic classes defined in (f). Red: numbers of proteins belonging to the RNA interactome. Blue: numbers of proteins not belonging to the interactome.
Figure 5
Figure 5. Possible explanations for dynamic behaviour of some of the RBPs during the MZT.
(a) Ten most enriched GO terms related to biological process among the class 3 RBPs and RBP candidates (‘dynamic binders'). (b) Log2 change in expression levels (RPKM) of putative transcript targets of four splicing factors and all genes. Splicing factors' targets are identified as transcripts whose abundance is significantly affected (|z-score|>2) upon depletion of a splicing factor. Comparison of changes in expression of all genes and splicing factor targets using the Student t-test resulted in the following P values: ASF/SF2–0.6698; Hrp48–0.1532; PSI—0.5239; B52–0.1847. (c) Enrichment of alternatively spliced mRNAs in the whole RNA interactome and in the three dynamic classes. Odds ratios are presented. (d) Pfam domains enriched among exons that are differentially used between 0–1 h and 4.5–5.5 h embryos Known RBDs are indicated in red.

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