An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis
2020; Elsevier BV; Volume: 19; Issue: 9 Linguagem: Inglês
10.1074/mcp.ra119.001912
ISSN1535-9484
AutoresPrashali Bansal, Johannes Madlung, Kristina Schaaf, Boris Maček, F. Bono,
Tópico(s)RNA and protein synthesis mechanisms
ResumoDuring Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans. During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans. The post-transcriptional regulation of gene expression requires several trans-acting factors that regulate the life cycle of an mRNA (1Moore M.J. Proudfoot N.J. Pre-mRNA Processing Reaches Back toTranscription and Ahead to Translation.Cell. 2009; 136: 688-700Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Many of these factors are RNA-binding proteins (RBPs) that interact with the maturing mRNAs to form functional messenger ribonucleoprotein complexes (mRNPs), interconnecting various steps of RNA metabolism, thereby controlling gene expression (1Moore M.J. Proudfoot N.J. 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Genetics of nanos Localization in Drosophila.Dev. Dyn. 1994; 199: 103-115Crossref PubMed Google Scholar). In addition to oogenic processes, Nos and Stau are also involved in the development of the Drosophila nervous system (63Heraud-Farlow J.E. Kiebler M.A. The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity.Trends Neurosci. 2014; 37: 470-479Abstract Full Text Full Text PDF PubMed Google Scholar, 64De Keuckelaere E. Hulpiau P. Saeys Y. Berx G. van Roy F. Nanos genes and their role in development and beyond.Cell. Mol. Life Sci. 2018; 75: 1929-1946Crossref PubMed Scopus (9) Google Scholar). Many RBPs in Drosophila oogenesis have overlapping functions that are likely differentially regulated. Little is known about this regulation and it may involve several as yet unidentified mRNP components. To comprehensively identify RBP interactors, we carried out a systematic in vivo purification screen of GFP-tagged RBPs coupled with MS. We employed both labeled and label-free MS methods and identified several proteins significantly enriched with the purified RBPs. The interactomes of the individual RBPs were largely independent with some overlap. Our screen identified several previously unknown interactions, many of which we validated in vitro. This work presents an extended interaction network of RBPs in Drosophila, offering a new reference point for future functional and structural studies of mRNA localization. For cloning purposes, total RNA was extracted from WT ovaries using the TRI-Reagent (Sigma), according to the manufacturer's instructions. RNA was reverse transcribed using Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (Thermo Fisher Scientific), in the presence of oligo (dT)15 primers. To express proteins in human HEK293 cells, genes of interest were amplified from Drosophila cDNA, or in some cases from the Drosophila Genomics Resource Center (DGRC) clones, using standard PCR conditions. Accession numbers of all the genes cloned are provided in supplemental Table S1. Fragments were cloned into mammalian expression vectors based on pEGFP-C1 (CLONTECH), bearing an N-terminal EGFP tag or modified to contain either HA or HA-Flag tags (provided by Elisa Izaurralde, MPI Tübingen). Full-length cDNAs were cloned, except for the protein Nucampholin (Ncm), where a sequence encoding amino acids 359-664 was amplified. The boundaries were designed based on the MIF4G domain of the human ortholog CWC22, which has been shown to bind eIF4AIII (65Buchwald G. Schüssler S. Basquin C. Le Hir H. Conti E. Crystal structure of the human eIF4AIII–CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E4611-E4618Crossref PubMed Scopus (0) Google Scholar). To serve as a control, either MBP or EGFP alone was used. All flies were kept at room temperature on standard Drosophila medium. Oregon R flies were used as WT. Fosmid lines expressing GFP-tagged proteins (66Sarov M. Barz C. Jambor H. Hein M.Y. Schmied C. Suchold D. Stender B. Janosch S. K J V.V. Krishnan R.T. Krishnamoorthy A. Ferreira I.R.S. Ejsmont R.K. Finkl K. Hasse S. Kämpfer P. Plewka N. Vinis E. Schloissnig S. Knust E. Hartenstein V. Mann M. Ramaswami M. VijayRaghavan K. Tomancak P. Schnorrer F. A genome-wide resource for the analysis of protein localisation in Drosophila.Elife. 2016; 5: e12068Crossref PubMed Scopus (124) Google Scholar) were purchased from the Vienna Drosophila Resource Center (VDRC 318283, 318719, 318195, 318157, 318898, 318766). To generate the control fly-line expressing the tag only, the tag sequence was cloned in a modified pUAST-attB vector (67Lazzaretti D. Veith K. Kramer K. Basquin C. Urlaub H. Irion U. Bono F. The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease.Nat. Struct. Mol. Biol. 2016; 23: 705-713Crossref PubMed Scopus (6) Google Scholar) (without UAS sites or SV40 poly(A) signal), downstream of a moderately expressing exu promoter using KpnI and BamHI sites. The purified vector was injected into embryos from a recombinant stock with a genotype y[1] M{vas-int.Dm} ZH-2A w[*]; PBac{y[+]-attP-3B}VK00033 (BDSC 24871). Transgenic flies were identified in the F1 generation by the presence of red eyes (dsRed) and a stable fly line was established. Human HEK293 cells were grown at 37 °C in the presence of 5% CO2 in standard Dulbecco's Modified Eagle Medium, supplemented with 10% heat-inactivated fetal bovine serum, Glutamine and Penicillin-Streptomycin solution. For co-immunoprecipitations (co-IP), transfections were carried out in six-well plates with Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendations. Typically, 5 μg of DNA was transfected in each well and the ratio of two plasmids was adjusted based on their expression levels. If required, a third empty plasmid with HA tag was supplemented, to reach a total amount of 5 μg. Cells were collected 2 days after transfection and washed with PBS before lysis. Cells were lysed for 15 min on ice in a buffer containing 50 mm Tris-HCl pH 7.5 at 4 °C, 100 mm NaCl, 250 mm Sucrose, 0.1% Nonidet P-40, 1 mm DTT, supplemented with protease inhibitors (cOmplete™, EDTA-free Protease inhibitor mixture, Roche). For efficient lysis, cells were mechanically sheared by passing them through a needle (Sterican 21G 7/8” Ø 0.8X22mm) several times. Cell lysates were cleared at 16,000 × g for 15 min at 4 °C and supernatants were incubated with 5 μl/ml of RNase A/T1 (Thermo Fisher Scientific) for 30 min at 4 °C. After clearing the lysate again at 16,000 × g for 15 min, 12-20 μl of GFP-TRAP MA beads (Chromotek) were added to the supernatant and the mixtures were rotated for 1 h at 4 °C. For Flag pull-downs, 1.8 μg of monoclonal anti-Flag antibody (Sigma #F1804) was added to the supernatant, after the RNase treatment. After 1 h at 4 °C in rotation, 20 μl of GammaBind Plus Sepharose beads (GE Healthcare) were added, and the mixtures were rotated for an additional hour at 4 °C. Beads were washed with lysis buffer and proteins were eluted in sample buffer by boiling at 95 °C for 10 min. Ovaries from well-fed flies were dissected in PBS and stored at −80 °C. For immunoprecipitation, frozen ovaries were thawed on ice in lysis buffer (50 mm Tris-HCl pH 7.5 at 4 °C, 100 mm NaCl, 250 mm Sucrose, 0.1% Nonidet P-40 and 1 mm DTT) and pooled together in required numbers (see supplemental Table S2). Ovaries were homogenized with a glass pestle in a tissue homogenizer in lysis buffer (320 μl/40 flies) supplemented with protease inhibitors (cOmplete™, EDTA-free Protease inhibitor mixture, Roche). Lysates were cleared by centrifugation at 21,000 × g for 20 min at 4 °C and 5 μl/ml of RNase A/T1 (Thermo Fisher Scientific) was added to the supernatants. After incubation at 4 °C for 30 min, lysates were cleared again and 30–60 μl of GFP-TRAP MA beads (Chromotek, Planegg-Martinsried, Germany) were added. The mixtures were incubated for 1 h at 4 °C in rotation. Beads were washed with lysis buffer and proteins were eluted as described above. Eluates were separated on 10% polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were blocked in PBS containing 5% milk powder and 0.1% Tween-20. HA-tagged, HA-Flag-tagged and GFP-tagged proteins were detected using HRP-conjugated monoclonal anti-HA (1:5000, BioLegend #901501) or polyclonal anti-GFP antibodies (1:2000, Thermo Fisher Scientific #A11122) respectively. Blots were developed with ECL (GE Healthcare) reagents, as recommended by the manufacturer, and imaged using an Amersham Pharmacia Biotech Imager 600 (GE Healthcare). The raw immunoblots are shown in supplemental data S6. For analysis of proteins interacting with each tagged RBP, both label-free and dimethyl labeling MS experiments were performed and raw data were processed by the MaxQuant software as described below. Proteome data comprised a total of 21 raw files (3 biological replicates from each sample) for label-free MS and 2 raw files (2 biological replicates from each sample) for dimethyl labeling MS. Tag alone was used as a negative control for both analyses. For proteome measurements, eluates were separated on a NuPAGE Bis-Tris precast 4-12% gradient gel (Invitrogen). Samples were run ∼2 cm into the gel and bands were visualized with a 0.1% Colloidal Coomassie Blue st
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