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In Vivo Stable Isotope Labeling of Fruit Flies Reveals Post-transcriptional Regulation in the Maternal-to-zygotic Transition

2009; Elsevier BV; Volume: 8; Issue: 7 Linguagem: Inglês

10.1074/mcp.m900114-mcp200

1535-9484

Joost W. Gouw, Martijn W. H. Pinkse, Harmjan R. Vos, Yuri M. Moshkin, C. Peter Verrijzer, Albert J. R. Heck, Jeroen Krijgsveld,

Physiological and biochemical adaptations

An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that ∼500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development. An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that ∼500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development. In many organisms, the first few hours of development are controlled by maternal proteins and mRNAs, which are deposited into the egg during oogenesis. After fertilization, the primary roles of these factors are to facilitate zygotic transcription and to establish the initial body framework. In Drosophila, zygotic transcription is initiated after approximately 2 h of development when the first 13 synchronous nuclear divisions give rise to the formation of the syncytial blastoderm. This is also referred to as the maternal-to-zygotic transition (MZT). 1The abbreviations used are:MZTmaternal-to-zygotic transitionSCXstrong cation exchangeFPfalse-positiveCVcoefficient of variationGOgene ontologymiRNAmicro-RNAPABPpoly(A)-binding proteinSMGSmaugCP1cysteine proteinase-1XICextracted ion chromatogramEASEExpression Analysis Systematic ExplorerOSKOskarME31Bmaternal expression at 31BYPSYpsilon Schachtel The first developmental processes controlled by zygotic factors are mitotic cycle 14 and the cellularization of the blastoderm thereby hallmarking the midblastula transition. Although the existence of this process has been known for a long time (1Wieschaus E. Gehring W. Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster.Dev. Biol. 1976; 50: 249-263Crossref PubMed Scopus (186) Google Scholar, 2Zalokar M. Autoradiographic study of protein and RNA formation during early development of Drosophila eggs.Dev. 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Parameters controlling transcriptional activation during early Drosophila development.Cell. 1986; 44: 871-877Abstract Full Text PDF PubMed Scopus (276) Google Scholar), the molecular mechanisms regulating the transition from mother to zygote are only beginning to be unraveled. maternal-to-zygotic transition strong cation exchange false-positive coefficient of variation gene ontology micro-RNA poly(A)-binding protein Smaug cysteine proteinase-1 extracted ion chromatogram Expression Analysis Systematic Explorer Oskar maternal expression at 31B Ypsilon Schachtel The mother transfers a large number of mRNAs to the oocyte, estimated at ∼7,000 transcripts (6De Renzis S. Elemento O. Tavazoie S. Wieschaus E.F. Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo.PLoS Biol. 2007; 5: e117Crossref PubMed Scopus (207) Google Scholar, 7Tadros W. Goldman A.L. Babak T. Menzies F. Vardy L. Orr-Weaver T. Hughes T.R. Westwood J.T. Smibert C.A. Lipshitz H.D. SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its translation is activated by the PAN GU kinase.Dev. Cell. 2007; 12: 143-155Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The bulk of these are degraded (8Bashirullah A. Halsell S.R. Cooperstock R.L. Kloc M. Karaiskakis A. Fisher W.W. Fu W. Hamilton J.K. Etkin L.D. Lipshitz H.D. Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster.EMBO J. 1999; 18: 2610-2620Crossref PubMed Scopus (177) Google Scholar), whereas a selected set needs to be stabilized allowing translation to sustain development of the embryo. This is achieved by the combined effects of mRNA localization, (de)stabilization by maternal and zygotic proteins, and translational repression and activation. Over the years it has become clear that in Drosophila multiple mechanisms act simultaneously to achieve protein expression at the right dose, at the right time, and at the right location. One way of localizing a particular protein is to stabilize and localize its mRNA transcript prior to translation, ensuring high levels of protein to restricted well defined cytoplasmic positions (9Kloc M. Etkin L.D. RNA localization mechanisms in oocytes.J. Cell Sci. 2005; 118: 269-282Crossref PubMed Scopus (116) Google Scholar, 10St Johnston D. Moving messages: the intracellular localization of mRNAs.Nat. Rev. Mol. Cell Biol. 2005; 6: 363-375Crossref PubMed Scopus (446) Google Scholar). This complements mechanisms suppressing activation of untranslated transcripts, which have been shown to aggregate in specific cytoplasmic granules known as P bodies (11Eulalio A. Behm-Ansmant I. Izaurralde E. P bodies: at the crossroads of post-transcriptional pathways.Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (751) Google Scholar, 12Parker R. Sheth U. P bodies and the control of mRNA translation and degradation.Mol. Cell. 2007; 25: 635-646Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). One of the important questions in the activation of the zygotic genome relates to the origin of proteins either by deposition in the oocyte by the mother or by transcriptional and translational activity in the embryo. Although recent proteomics studies aimed to define the Drosophila proteome (13Gong L. Puri M. Unlü M. Young M. Robertson K. Viswanathan S. Krishnaswamy A. Dowd S.R. Minden J.S. Drosophila ventral furrow morphogenesis: a proteomic analysis.Development. 2004; 131: 643-656Crossref PubMed Scopus (55) Google Scholar, 14Brunner E. Ahrens C.H. Mohanty S. Baetschmann H. Loevenich S. Potthast F. Deutsch E.W. Panse C. de Lichtenberg U. Rinner O. Lee H. Pedrioli P.G. Malmstrom J. Koehler K. Schrimpf S. Krijgsveld J. Kregenow F. Heck A.J. Hafen E. Schlapbach R. Aebersold R. A high-quality catalog of the Drosophila melanogaster proteome.Nat. Biotechnol. 2007; 25: 576-583Crossref PubMed Scopus (233) Google Scholar, 15Zhai B. Villén J. Beausoleil S.A. Mintseris J. Gygi S.P. Phosphoproteome analysis of Drosophila melanogaster embryos.J. Proteome Res. 2008; 7: 1675-1682Crossref PubMed Scopus (230) Google Scholar), they investigated a different developmental event, or they did not specifically focus on fly development. In a number of recent studies genomics techniques were used to distinguish maternal from zygotic gene expression. Lécuyer et al. (25Lécuyer E. Yoshida H. Parthasarathy N. Alm C. Babak T. Cerovina T. Hughes T.R. Tomancak P. Krause H.M. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.Cell. 2007; 131: 174-187Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar) used high resolution fluorescent in situ hybridization assuming that maternal and zygotic transcripts localize in the cytoplasm and nucleus, respectively. De Renzis et al. (6De Renzis S. Elemento O. Tavazoie S. Wieschaus E.F. Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo.PLoS Biol. 2007; 5: e117Crossref PubMed Scopus (207) Google Scholar) addressed a similar question by investigating chromosomeablated mutants to discriminate between transcriptional and post-transcriptional regulation of gene expression, and it was estimated that ∼20% of the transcripts at cycle 14 were of zygotic origin. Although the presence and precise localization of transcripts are crucial to understand developmental activation of the embryo, they do not necessarily allow extrapolation to protein expression. Notably multiple mechanisms shown to determine mRNA stability and translational activity (e.g. dependent on or independent of deadenylation, targets of RNA silencing, or transacting factors) provide an additional level of regulation (16Vardy L. Orr-Weaver T.L. Regulating translation of maternal messages: multiple repression mechanisms.Trends Cell Biol. 2007; 17: 547-554Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The result of the combined effect of these post-transcriptional processes can only be captured by determining expression levels of individual proteins before and after MZT. Therefore, we used a proteomics approach quantifying the relative protein expression levels before (1.5 h after oviposition, embryonic stages 1–3) and after MZT (4.5 h after oviposition, embryonic stages 6–9). By applying a combined approach using in vivo labeling of fruit flies by the incorporation of stable isotope-coded nitrogen (15N) (17Krijgsveld J. Ketting R.F. Mahmoudi T. Johansen J. Artal-Sanz M. Verrijzer C.P. Plasterk R.H. Heck A.J. Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.Nat. Biotechnol. 2003; 21: 927-931Crossref PubMed Scopus (345) Google Scholar) combined with LC-MS/MS, more than 1,700 proteins could be quantitated in two biological independent experiments. About half of these changed in abundance of which ∼350 proteins increased, providing for the first time direct evidence of the identity of proteins as a product of embryonic translation in a large scale approach. Although these up-regulated proteins represent a wide variety of functional classes, maternal proteins were among the most dramatically down-regulated proteins including transacting factors involved in regulation of mRNA stability (including maternal expression at 31B (ME31B), Smaug (SMG), and a number of proteins interacting with these). Moreover specific down-regulation of these proteins appears to be governed by a post-transcriptional mechanism as evidenced by direct comparison of protein and transcript levels in the same samples. In addition, evidence was found that a limited number of proteins, including poly(A)-binding protein (PABP) and cysteine proteinase-1 (CP1), were subject to post-translational processing leading to truncation, possibly resulting in an altered function of these proteins. Altogether this study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin associated with embryonic development in Drosophila. Wild-type OregonR flies were maintained by standard methods at 25 °C and were labeled as described previously (17Krijgsveld J. Ketting R.F. Mahmoudi T. Johansen J. Artal-Sanz M. Verrijzer C.P. Plasterk R.H. Heck A.J. Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.Nat. Biotechnol. 2003; 21: 927-931Crossref PubMed Scopus (345) Google Scholar). Briefly larvae were grown in boxes containing 15N-labeled or unlabeled yeast and kept at 25 °C throughout larval and pupal developmental stages. Hatched flies were transferred to fly cages and kept on 15N-labeled or unlabeled yeast. Embryos were collected on agarose-agar plates completed with a small amount of 15N-labeled or unlabeled yeast that were removed from the fly cage after 90 min. Unlabeled stage 6–9 embryos were obtained by aging the 0–90-min embryos using standard methods for another 180 min, whereas 15N-labeled embryos were processed immediately. Embryos were washed in water and dechorionated by incubation in 2.5% sodium hypochlorite for 90 s followed by another wash and then kept at −20 °C. A biological duplicate, independent experiment was performed with swapped labels (i.e. 0–90 min unlabeled and 180–270 min 15N-labeled). Equal amounts of labeled and unlabeled embryos were combined and lysed in 8 m urea and 50 mm ammonium bicarbonate. Cellular debris were pelleted by centrifugation at 20,000 × g for 20 min. Prior to digestion, proteins were reduced with 1 mm DTT and alkylated with 2 mm iodoacetamide. The mixture was diluted 4-fold to 2 m urea using 250 µl of 50 mm ammonium bicarbonate and 50 µl of 0.1 mg/ml trypsin solution and incubated overnight at 37 °C. Strong cation exchange was performed using a Zorbax BioSCX-Series II column (0.8-mm inner diameter × 50-mm length, 3.5 µm), a FAMOS autosampler (LC Packings, Amsterdam, The Netherlands), and a Shimadzu LC-9A binary pump and a SPD-6A UV detector (Shimadzu, Tokyo, Japan). Prior to strong cation exchange (SCX) chromatography, protein digests were desalted using a small plug of C18 material (3M Empore C18 extraction disk) packed into a GELoader tip (Eppendorf) similar to what has been described previously (18Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1833) Google Scholar) onto which ∼10 µl of Aqua C18 (5 µm, 200 Å; Phenomenex) material was placed. The eluate was dried completely and subsequently reconstituted in 20% acetonitrile and 0.05% formic acid. After injection, a linear gradient of 1% min−1 solvent B (500 mm KCl in 20% acetonitrile and 0.05% formic acid, pH 3.0) was performed. A total of 28 SCX fractions (1 min each, i.e. 50-µl elution volume) were manually collected and dried in a vacuum centrifuge. Dried residues were reconstituted in 50 µl of 0.1 m acetic acid and were analyzed by nanoflow liquid chromatography using an Agilent 1100 HPLC system (Agilent Technologies) coupled on line to a 7-tesla LTQ-FT-ICR mass spectrometer (Thermo Electron). The liquid chromatography part of the system was operated in a setup essentially as described previously (19Meiring H.D. van der Heeft E. ten Hove G.J. de Jong A.P. Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis.J. Sep. Sci. 2002; 25: 557-568Crossref Scopus (226) Google Scholar). Aqua C18, 5-µm (Phenomenex) resin was used for the trap column, and ReproSil-Pur C18-AQ 3-µm (Dr. Maisch GmbH) resin was used for the analytical column. Peptides were trapped at 5 µl/min in 100% solvent A (0.1 m acetic acid in water) on a 2-cm trap column (100-µm inner diameter, packed in house) and eluted to a 40-cm analytical column (50-µm inner diameter, packed in house) at ∼100 nl/min in a 150-min gradient from 10 to 40% solvent B (0.1 m acetic acid in 8:2 (v/v) acetonitrile/water). The eluent was sprayed via standard coated emitter tips (New Objective) butt-connected to the analytical column. The mass spectrometer was operated in datadependent mode, automatically switching between MS and MS/MS. Full scan mass spectra (from m/z 300 to 1,500) were acquired in the FT-ICR cell with a resolution of 100,000 at m/z 400 after accumulation to a target value of 500,000. The three most intense ions at a threshold above 5,000 were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35% after accumulation to a target value of 15,000. All MS2 spectra were converted to single DTA files using Bioworks 3.1 (Thermo) with default parameters and merged into a Mascot generic format file that was searched twice to identify both unlabeled and 15N-labeled peptides using an in-house licensed Mascot v2.1.0 search engine (Matrix Science) against a concatenated database containing both forward and reversed entries from an Integr8 D. melanogaster database (version 20060806) consisting of 32,508 sequences. Carbamidomethylcysteine was set as a fixed modification; oxidized methionine, protein N-acetylation, and N-terminal pyroglutamate were set as variable modifications. Trypsin was specified as the proteolytic enzyme, and up to two missed cleavages were allowed. The mass tolerance of the precursor ion was set to 15 ppm, and that of fragment ions was set to 0.8 Da. Both search results (14N and 15N identifications) were merged into one HTML result page using an in-house developed Perl script for qualitative and quantitative analysis. A false-positive discovery rate of 8 were selected. Labeling, hybridization, washes, and staining of microarrays were performed according to Affymetrix specifications. Statistical analysis of the microarray data was performed using R and Bioconductor free software as described previously (23Moshkin Y.M. Mohrmann L. van Ijcken W.F. Verrijzer C.P. Functional differentiation of SWI/SNF remodelers in transcription and cell cycle control.Mol. Cell. Biol. 2007; 27: 651-661Crossref PubMed Scopus (105) Google Scholar). Gene expression indexes were calculated using the robust multichip average algorithm implemented in the Bioconductor affy package. Distribution of the expression indexes is bimodal; therefore we applied the multiple covariance determinant algorithm implemented in the rrcov R package to filter non-expressing genes. 4,657 genes were selected for further analysis. Details of the statistical analysis and R scripts will be provided upon request. About 2 mg of early and late embryos and 18 mg of adult flies were lysed in buffer containing 30 mm Tris, 7 m urea, 2 m thiourea, 50 mm DTT, and 54 mm CHAPS and sonicated for 30 s. Lysates were centrifuged at 14,000 × g to pellet cellular debris. Protein concentration was determined by a 2D-Quant kit (Amersham Biosciences/GE Healthcare) using the standard protocol. For each experiment, 15 µg of protein was used as starting material, subjected to SDS-PAGE, and blotted. After transfer, the membrane was stained with Ponceau red to verify equal loading of the lysates. Subsequently the nitrocellulose membrane was blocked with 5% Protifar plus (Nutricia) and then incubated with antibodies against PABP, Rack1, and eIF3-S9 followed by a horseradish peroxidase-conjugated secondary antibody. The membranes were subjected to detection by enhanced chemiluminescence. Membranes were subsequently stripped at 50 °C in buffer containing 62.5 mm Tris, 2% SDS, and 0.1 m β-mercaptoethanol; blocked with 5% Protifar plus; and then incubated with anti-α-tubulin antibody followed by the same procedure as described above. The strategy used to identify and differentially quantify proteins in embryos in Browne's stages 1–3 and 6–9 is shown schematically in Fig. 1. We started our approach with ∼10 ml of 15N-labeled and unlabeled flies and collected "heavy" and "light" embryos for 90 min. Light embryos were allowed to develop for another 180 min to reach stage 6–9. A second biological independent experiment with reversed labels ("label swap") was conducted as well. Embryos were visually inspected after harvesting to confirm their intended developmental stage (supplemental Fig. 1, A–C). Incorporation of 15N was verified by mass spectrometric analysis of labeled embryos (supplemental Fig. 1D). Labeled and unlabeled samples were combined (Fig. 1A), proteins were extracted, digested using trypsin, and subjected to SCX as the first separation step (Fig. 1B). One-minute SCX fractions were collected with peptides eluting in fractions 5–33. Each of these 28 fractions was subjected to the second dimension nano-LC MS/MS. A typical chromatogram of one fraction is shown in Fig. 1C. Extended column length (40 cm) and gradient times (2.5 h) were used to obtain optimal peptide separation over more than 2 h (Fig. 1C). Analysis of these fractions led to an average of 10,000 fragment spectra per SCX fraction. Altogether more than 250,000 spectra were searched against a Drosophila protein database consisting of "forward" and "reversed" protein sequences for protein identifications and an estimation of the false-positive (FP) discovery rate. To determine an optimal FP rate, peptide scores from both parts of the database were plotted (supplemental Fig. 2). With a peptide cutoff score of 20 (horizontal line in supplemental Fig. 2), a peptide FP rate of ∼4% resulted in 79,196 peptide identifications. In addition to the peptide cutoff score, we used a minimum of two peptides per protein and a protein cutoff score of 60. This led to 2,736 protein identifications with a false-positive discovery rate of <1%. The complete list of all proteins identified in stages 1–3 as well as stages 6–9 is given in supplemental Table 1. Subsequently proteins were quantified by integrating all MS peak areas of identified peptides using an in-house adapted version of MSQuant (24Schulze W.X. Mann M. A novel proteomic screen for peptide-protein interactions.J. Biol. Chem. 2004; 279: 10756-10764Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) (Fig. 1C). Using a minimum of two quantifiable peptides per protein, a total of 2,232 proteins were quantified (Fig. 1D). Peptides that could not be quantified represent mainly low abundance peptides that disappear in the noise or produce an insufficient number of data points for proper integration (each peptide was manually verified). Qualitative as well as quantitative data were parsed into a searchable PostGreSQL database (Fig. 1D) (available upon requests). Protein identifications were obtained by stringent filtering criteria, and therefore the compendium of the proteins found in stages 1–3 and 6–9 (supplemental Table 1) provides a valuable resource for further exploration. To accurately assess the quantitative difference between early and late embryos, thereby uncovering proteins associated with the maternal-to-zygotic transition, protein ratios were based on the quantitative results of two biological independent experiments where labels were swapped (unlabeled early embryos and 15N-labeled late embryos). A total of 1,737 proteins were quantified in two experiments. Fig. 2A shows a scatter plot of the protein ratios of both experiments where a correlation coefficient of 0.831 indicates that protein ratios are in good agreement. An overview of the relative expression patterns of these 1,737 proteins is shown in Fig. 2B including error bars that represent the S.D. between the two protein ratios. Protein quantitation was of high quality, reflected by the small average error in each of the two individual data sets (that is, the error between the peptides of the same protein) of 18 and 11%, respectively. Furthermore the average variation between proteins quantitated in both data sets was 11%. Proteins identified in only one replicate most probably suffered from undersampling during mass spectrometric data acquisition, but given the high correlation between the replicates and the small standard deviations, most of them are likely to be plausible. Yet to increase confidence, only the proteins that were quantitated in both data sets were taken for further analysis. The accuracy of protein quantitation in our approach was evidenced further by the abundance ratios of ribosomal proteins. Among the expression ratios of the 125 subunits identified in this study, a remarkable observation can be made i