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Combined Use of RNAi and Quantitative Proteomics to Study Gene Function in Drosophila
Abstract
RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 10-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high for those mRNAs that changed significantly by microarray.
... proved when limiting the dataset to include only transcripts that changed significantly (53). These results suggest that although RNA abundance is only a moderate predictor of protein abundance, changes in RNA abundance are often good indicators of changes in protein abundance. ...
... With over 15,000 genes in the Chlamydomonas genome and ϳ12,300 soluble proteins estimated (using TMHMM), only 23% of all gene products were detected and quantified. Our transcript-protein correlation estimates agree with other studies of other organisms ranging from E. coli and yeast to mouse and human (52,53,(77)(78)(79)(80). Our analysis also suggests that this overall correlation does not change depending on the nutritional state. ...
... In other words, changes in RNA abundance can serve as a reliable predictor of changes at the protein level. Studies on yeast, corn, and Drosophila further support this notion (53,81,82). ...
Trace metals such as copper, iron, zinc and manganese play important roles in several biochemical processes including respiration and photosynthesis. Using a label-free, quantitative proteomics strategy (MSE), we examined the impact of deficiencies of these micronutrients on the soluble proteome of Chlamydomonas reinhardtii. We quantified 1000 proteins with abundances within a dynamic range of 3-4 orders of magnitude and demonstrated statistically significant changes in approximately 200 proteins in each metal deficient growth condition relative to nutrient replete media. From analysis of the Pearson's coefficient we also examined the correlation between protein abundance and transcript abundance (as determined by RNA-Seq analysis) and found moderate correlations under all nutritional states. Interestingly, the correlation between the change in protein abundance and the change in transcript abundance in cells grown in metal replete and metal deficient conditions is even stronger. Examples of new discoveries highlighted in this work include the accumulation of O2 labile, anaerobiosis-related enzymes (Hyd1, Pfr1, and Hcp2) in copper deficient cells, loss of various stromal and lumenal, photosynthesis-related proteins including plastocyanin in iron limited cells, large accumulation (from undetectable amounts to over 1,000 zmol/cell) of two COG0523 domain-containing proteins in zinc deficient cells, and the preservation of photosynthesis proteins in manganese deficient cells despite known losses in photosynthetic function in this condition.
... Selecting differential candidates for transcriptome| proteome comparison Poor to moderate transcriptome|proteome correlation has been shown previously (Bonaldi et al. 2008). Remarkably, the authors found a much better correlation when they plotted the level ratios of those candidates showing a significant difference between two conditions. ...
... implying poor to moderate transcript|protein correlation (Fig. 3). (iii) A better correlation, although less than expected (Bonaldi et al. 2008), occurred when viewing significant inter-region ratios only (0.53-0.67; except for CN/SOC: 0.06; Fig. 6). ...
A correlation between the transcriptome and the proteome strongly depends on the sample and the experimental condition. The brain has a more complex set of proteins than other organs. Moreover, its proteome is highly regulated by various types of post-translational modifications, such as phosphorylation, ubiquitination, glycosylation, and oxidation. The degree of translational and post-translational regulation affects the degree of the transcriptome-proteome correlation. The asymmetric morphology of neurons can be a further cause of non-correlating gene products in the brain: proteins may be spatially separated from their transcripts, e.g. by axonal transport. We expect that these factors – and thus the transcriptome-proteome correlation – vary between brain regions. To address this issue, we here analyzed the highly specialized cochlear nuclear complex (CN), superior olivary complex (SOC), and inferior colliculus (IC) in the rat auditory brainstem. As a fourth and more general region, we included the rest of the brain (Rest). We compared these brain regions to each other regarding their transcriptome and their proteome, enclosed by microarray analysis and lable-free mass spectrometry, respectively. The protein ratios clearly correlated with the corresponding transcript ratios when comparing CN vs. Rest, SOC vs. Rest, and IC vs. Rest (R2: 0.25-0.39). A smaller degree of correlation emerged when comparing CN vs. IC and SOC vs. IC (R2: 0.15-0.17). Interestingly, no transcriptome-proteome correlation was detected when comparing CN vs. SOC (R2: 0.04). Further, region-typical proteins overlapped only partially with region-typical transcripts. The smallest overlap was found in the groups of SOC-typical and -atypical gene products: among 30 SOC-typical proteins and 7 SOC-typical transcripts, only 1 (Slc6a5 = GlyT2) intersected. Together, our findings emphasize the importance of proteomic research, especially as there are structures (e.g. the SOC) which lack a clear correlation between transcripts and corresponding proteins.
... Targeting a gene via RNAi is often used to study its function.However, the reports on the global changes of mRNA and protein profile that occur after RNAi targeting of a specific gene have been limited 21,22 . For example, after knock down of ISWI in Drosophila SL2 cells, Bonaldi et al. studied the global changes of transcriptome and proteome using microarray and SILAC-MS technologies, and found that there is a very little correlation between mRNA and protein level 23 . In cultured male-like tissue cells of D. melanogaster, the expression level of many genes on the X chromosome decreased after RNAi targeting MSL complex; however, the gene expression on autosomes remained largely unchanged 24 . ...
... We identified 12 genes in which 5 were up-regulated and 7 were down-regulated at both mRNA and protein level (Supplementary Table 2). Similar to the previous report 23 , the fold of change in mRNA and protein levels was different. Recently, accumulated evidences showed that the TOR pathway regulates protein synthesis and cell proliferation through integrating multiple upstream signals 25 . ...
The target of rapamycin (TOR) positively controls cell growth in response to nutrients such as amino acids. However, research on the specific nutrients sensed by TOR is limited. Glutamine (Gln), a particularly important amino acid involved in metabolism in organisms, is synthesised and catalysed exclusively by glutamine synthetase (GS), and our previous studies have shown that Gln may regulate fecundity in vivo levels of the brown planthopper (BPH) Nilaparvata lugens. Until now, it has remained unclear whether Gln activates or inhibits the TOR signalling pathway. Here, we performed the combined analyses of iTRAQ (isobaric tags for relative and absolute quantification) and DGE (tag-based digital gene expression) data in N.lugens at the protein and transcript levels after GS RNAi, and we found that 52 pathways overlap, including the TOR pathway. We further experimentally demonstrate that Gln activates the TOR pathway by promoting the serine/threonine protein kinase AKT and inhibiting the 5′AMP-activated protein kinase AMPK phosphorylation activity in the pest. Furthermore, TOR regulates the fecundity of N. lugens probably by mediating vitellogenin (Vg) expression. This work is the first report that Gln activates the TOR pathway in vivo.
... AB-GADPH, Glyceraldehyde 3-phosphate dehydrogenase; GADPH2, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase isoform; ACLB1, ACLB2, ATP citrate lyase a-subunit; CS, Citrate synthase; ENO1, ENO2, ENO3, Enolase; FBA2, FBA3, Fructose-bisphosphate aldolase; FRK2, FRK3, Fructokinase; GPI1, GPI2, Glucose-6-phosphate isomerase; MDH, malate dehydrogenase; PEPC1, PEPC2, PEPC3 phosphoenolpyruvate carboxylase; PEPCK2, phosphoenolpyruvate carboxykinase; PGM, Phosphoglucomutase; PK, pyruvate kinase; TPI, Triosephosphate isomerase; Susy1, Sucrose synthase; TKL, Transketolase 1, 1,3BPG, 1,3-Bisphosphoglycerate; RuBP, Ribulose-1,5-bisphosphate; R5P, ribulose-5-phosphate; FDB Fructose-1,6-Bisphosphate; F6P fructose-1,6-bisphosphate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate, DHAP, dihydroxyacetone phosphate. 36 and Drosophila melanogaster 37 . In the latter case, mRNA and protein levels showed a fairly good Pearson correlation coefficient of r = 0.8, when genes not responding to the applied stimulus were removed from the analysis. ...
The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.
... 2) Plotting transcript levels versus cognate protein levels revealed R 2 values of 0.34 to 0.54, implying poor to moderate transcript|protein correlation (Fig. 3). 3) Better correlation, although less than expected (Bonaldi et al. 2008), occurred when viewing significant inter-region ratios only (0.53 to 0.67; except for CN/SOC: 0.06; Fig. 6). ...
Transcription, translation, and turnover of transcripts and proteins are essential for cellular function. The contribution of those factors to protein levels is under debate, as transcript levels and cognate protein levels do not necessarily correlate due to regulation of translation and protein turnover. Here we propose neuronal polarity as a third factor that is particularly evident in the CNS, leading to considerable distances between somata and axon terminals. Consequently, transcript levels may negatively correlate with cognate protein levels in CNS regions, i.e., transcript and protein levels behave reciprocally. To test this hypothesis, we performed an integrative inter‐omics study and analyzed three interconnected rat auditory brainstem regions (cochlear nuclear complex, CN; superior olivary complex, SOC; inferior colliculus, IC) and the rest of the brain as a reference. We obtained transcript and protein sets in these regions of interest (ROIs) by DNA microarrays and label‐free mass spectrometry, and performed principal component and correlation analyses. We found 508 transcript|protein pairs and detected poor to moderate transcript|protein correlation in all ROIs, as evidenced by coefficients of determination from 0.34 to 0.54. We identified 57‐80 negatively correlating gene products in the ROIs and intensively analyzed four of them for which the correlation was poorest. Three cognate proteins (Slc6a11, Syngr1, Tppp) were synaptic and hence candidates for a negative correlation because of protein transport into axon terminals. Thus, we systematically analyzed the negatively correlating gene products. Gene ontology analyses revealed overrepresented transport/synapse‐related proteins, supporting our hypothesis. We present 30 synapse/transport‐related proteins with poor transcript|protein correlation. In conclusion, our analyses support that protein transport in polar cells is a third factor that influences the protein level and, thereby, the transcript|protein correlation.
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... The data and correlation analyses suggested that the quantitation values in our SILAC experiments are reliable and can be used for further analysis. Setting a twofold difference in expression level (Bonaldi et al. 2008) between the treated and the control as the cutoff revealed 45 dysregulated proteins (Table S1), including lipoprotein lipase (LPL) and lysozyme 1 (LYZ1) that were up-regulated and 43 proteins that were down-regulated by GO treatment (Figure 3(B,D)). ...
Although the toxicity and molecular mechanisms of graphene oxide (GO) have been reported for several cell types, no proteomic study of GO has yet been conducted on macrophage cells. In this study, we used proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) to quantify the proteomic changes in macrophage RAW 264.7 cells following GO treatment. We found 73 proteins that were significantly dysregulated after GO treatment. The down-regulated proteins included many ribosomal subunit proteins, indicating that GO affected cell growth. The most elevated proteins were lipoprotein lipase (LPL) and lysozyme 1 (LYZ1) which have not been reported before, and both can be used as candidate markers for GO exposure. Further enrichment analysis of the up-regulated proteins indicated these proteins are associated with the integrin complex and membrane rafts, as well as with two signal pathways: the phagosome and steroid biosynthesis pathways. We confirmed a GO concentration-dependent increase in membrane rafts and the production of phagosomes. GO exposure also induced necrotic cell death and an inflammation response in RAW 264.7 cells. We also observed an increase in the oxidative stress response (ROS) and autophagy, and the results suggest that ROS induced autophagy by the ROS-NRF2-P62 pathway.
... A principal vantagem oferecida por metodologias de marcação metabólica em relação às demais, e que justifica nossa escolha por SILAC, corresponde à ausência da influência da variabilidade técnica na quantificação, já que todas as etapas de preparação de amostra, espectrometria e análise são realizadas com as duas populações celulares simultaneamente.Nos resultados preliminares de proteômica quantitativa, cerca de 500 proteínas foram quantificadas em cada comparação knockdown/controle. Este valor é ainda baixo, já que em estudos de proteômica quantitativa utilizando SILAC, o número de proteínas quantificadas fica na casa dos milhares(BONALDI et al., 2008;DE GODOY et al.;. Além disso, o objetivo inicial deste estudo sempre foi quantificar os níveis de redução das proteínas STE7 após RNAi por espectrometria de massas, objetivo não alcançado neste experimento preliminar, já que as quinases STE7 não foram identificadas. ...
In Trypanosoma brucei, post-transcriptional mechanisms regulating gene expression are essential, especially those events that quickly modulate the adaptation to different environments. The signaling pathways of mitogen-activated protein kinases (MAPK) transduce environmental stimuli generating a range of intracellular responses. However, so far little is known about the function and relationship between the members of these pathways in T. brucei. Through the RNA interference (RNAi) approach combined with quantitative proteomics and phosphoproteomics, our study identified targets and pathways regulated by the five STE7 proteins (upstream activators of MAPK) and the impacts caused in the proteome and phosphoproteome after knockdown of these signaling components. Our results show that the knockdown of STE7-1, STE7-2, STE7-4 and STE7-5 decreased the growth of procyclic T. brucei in at least one of the tested conditions. Using SILAC-based mass spectrometry approach, we quantified differences in the levels of proteins and phosphorylation sites implicated in a range of biological functions, such as biosynthetic pathways, energy metabolism and translation. This study represents an important step towards the understanding of the role of these signaling components in a global context in Trypanosoma brucei.
... Because only ~ 10% of RNAs that show 2-fold or more change in transcriptional profiling or micro-arrays lead to alterations in protein levels (Bonaldi et al., 2008;Chick et al., 2016;de Sousa Abreu et al., 2009;Nie et al., 2007), we sought to directly measure the quantitative changes of proteins during these initiating events in direct reprogramming using multiplexed tandem mass tag (TMT 6-plex) mass spectrometry-based proteomics. This quantitative multiplexing approach increases throughput and minimizes variability because relative quantification is performed for up to six combined samples in a single analysis (Thompson et al., 2003). ...
Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications. Currently, application of this technology is limited by our lack of understanding of the molecular mechanisms that drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach, we identified the temporal global changes in protein abundance that occur during initial phases of iCM reprogramming. Collectively, our results show systematic and temporally distinct alterations in levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins. We have constructed protein relational networks associated with the initial transition of a fibroblast into an iCM. These findings demonstrate the presence of an orchestrated series of temporal steps associated with dynamic changes in protein abundance in a defined group of protein pathways during the initiating events of direct reprogramming.
... However, mRNAs are further translated into proteins, which perform the actual cellular functions. It has been shown in multiple species such as Saccharomyces cerevisiae (Griffin et al. 2002), Trypanosoma brucei (Butter et al. 2013), Caenorhabditis elegans (Grün et al. 2014), and human (Schwanhäusser et al. 2011), as well as in Drosophila melanogaster (Bonaldi et al. 2008), that transcript levels are only a moderate predictor for protein expression as they do not account for post-transcriptional processes such as translational regulation or protein stability (Vogel and Marcotte 2012;Liu et al. 2016). Recently, this has also been addressed with a developmental perspective in Caenorhabditis elegans (Grün et al. 2014), Xenopus laevis (Peshkin et al. 2015), and Trypanosoma brucei (Dejung et al. 2016), but not yet in Drosophila. ...
Drosophila melanogaster
is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling acrossDrosophila's life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogeneCyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface.
... When the information obtained from traditional prediction is limited, phenotypic profiling based on gene silencing can help to estimate the action mode of unknown genes [22]. Moreover, with the development of omics technology and bioinformatics, the global changes of mRNA and protein profiling that occur after knockdown of target genes can contribute to the construction of protein interaction network [6,23,24]. This means that functions of uncharacterized genes are more likely to be identified today. ...
... Furthermore, network analysis often led to the identification of potential targets that may be essential for understanding disease mechanism (Cantu et al., 2009;Chi et al., 2013). Reverse genetics approach using mutants, RNAi, and Crisper may target such host factors to silence the candidate gene/protein thereby imparting resistance (May et al., 2005;Bonaldi et al., 2008;Katiyar and Jin, 2010;Tinoco et al., 2010;Cary et al., 2011;Younis et al., 2014;Mishra et al., 2016). Targeting such host proteins for making the crop tolerant to Scleroninia infection may benefit breeding/quality improvement program preventing fruit spoilage during storage for post-harvest management of tomato (Jnra et al., 2000). ...
Fruit is an assimilator of metabolites, nutrients, and signaling molecules, thus considered as potential target for pathogen attack. In response to patho-stress, such as fungal invasion, plants reorganize their proteome, and reconfigure their physiology in the infected organ. This remodeling is coordinated by a poorly understood signal transduction network, hormonal cascades, and metabolite reallocation. The aim of the study was to explore organ-based proteomic alterations in the susceptibility of heterotrophic fruit to necrotrophic fungal attack. We conducted time-series protein profiling of Sclerotinia rolfsii invaded tomato (Solanum lycopersicum) fruit. The differential display of proteome revealed 216 patho-stress responsive proteins (PSRPs) that change their abundance by more than 2.5-fold. Mass spectrometric analyses led to the identification of 56 PSRPs presumably involved in disease progression; regulating diverse functions viz. metabolism, signaling, redox homeostasis, transport, stress-response, protein folding, modification and degradation, development. Metabolome study indicated differential regulation of organic acid, amino acids, and carbohydrates paralleling with the proteomics analysis. Further, we interrogated the proteome data using network analysis that identified two significant functional protein hubs centered around malate dehydrogenase, T-complex protein 1 subunit gamma, and ATP synthase beta. This study reports, for the first-time, kinetically controlled patho-stress responsive protein network during post-harvest storage in a sink tissue, particularly fruit and constitute the basis toward understanding the onset and context of disease signaling and metabolic pathway alterations. The network representation may facilitate the prioritization of candidate proteins for quality improvement in storage organ.
... Therefore, gene expression is rarely an accurate determinant of protein abundance [8]. Even though higher levels of correlation between gene and protein expression have been reported, [9] genes encoding histones [10] and ribosomal proteins [10,11] have been shown to correlate poorly with their respective proteins, due to the limitations of RNA-seq methodology. Additionally, when multiple Proteoform exist, each transcript would be assigned to the same gene, while proteomic tools would allow detection of each unique protein isoform, causing substantial discordances between data. ...
With the advances in high-throughput genome/transcriptome sequencing technologies and mass spectrometry (MS)-based proteomics, thousands of gene-protein pairs can be matched and merged in a single experiment. It is of interest to perform a correlative analysis of gene and protein expression data and investigate the nature of their similarity/dissimilarity as it could harbour potential biomarkers or drug targets. Manual determination of data points of interest quickly becomes a very complex and laborious process. Thus, there is a high demand for automated ‘omics’ data integration tools that can not only routinely match and combine gene and protein expression values but also provide a measure to highlight meaningful biological insights. In this work, we applied a fast and easy approach to integrate large proteomic and transcriptomic data derived from the deep analysis of glioma cancer stem cells (GSCs). The proposed algorithm provides a mathematical distance between two data sets and asignes a direction of their interrelation based on the abundancies. We distinguished three types of the data correlation: concordant, anticoncordant where protein abundance was higher than that of the corresponding RNA and anticoncordant where protein abundance was lower. We investigated the nature of the observed discordances and were able to separate different, phenotypically divergent, classes of GSC lines.
... To affinity-purify Su(s)-3XFLAG for MS analysis, 30 mL of log phase Su(s)-3XFLAG and control S2 cells were treated with 70 µM copper sulfate and grown for 24 h. The cells were harvested at a density of 1 × 10 7 cells/mL, and nuclear extracts were prepared as previously described by Bonaldi et al. (2008), except that SDS and deoxycholate were omitted from the buffer that was used to lyse the nuclei. The nuclear fraction was incubated with 75 µL bed volume of anti-FLAG magnetic beads (Sigma-Aldrich) for 2 h at 4°C and washed, pre-eluted with HA peptide, and eluted with the 3X-FLAG peptide as described in the Sigma-Aldrich protocol. ...
Although RNA polymerase II (Pol II) productively transcribes very long genes in vivo, transcription through extragenic sequences often terminates in the promoter-proximal region and the nascent RNA is degraded. Mechanisms that induce early termination and RNA degradation are not well understood in multicellular organisms. Here, we present evidence that the suppressor of sable [su(s)] regulatory pathway of Drosophila melanogaster plays a role in this process. We previously showed that Su(s) promotes exosome-mediated degradation of transcripts from endogenous repeated elements at an Hsp70 locus (Hsp70-αβ elements). In this report, we identify Wdr82 as a component of this process and show that it works with Su(s) to inhibit Pol II elongation through Hsp70-αβ elements. Furthermore, we show that the unstable transcripts produced during this process are polyadenylated at heterogeneous sites that lack canonical polyadenylation signals. We define two distinct regions that mediate this regulation. These results indicate that the Su(s) pathway promotes RNA degradation and transcription termination through a novel mechanism.
... Selbach and coworkers used this method first to identify interaction partners of ?-catenin and Cbl [147]. It has also been applied successfully in multiple myeloma cells for identification of interacting partners of 14-3-3 [148] and Stat3 [149], but also to investigate the role of BAG3 in human [150], and to study the molecular functions of Drosophila ISWI, an ATP-hydrolyzing motor present in different chromatin remodeling complexes [151]. ...
Stable isotope labeling by amino acids in cell culture (SILAC) is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein post-translational modifications (PTMs), to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Acquired MS raw data files from two biological replicates were processed with MaxQuant (version 1.3.0.5). 34 Enzyme specificity was set to allow for cleavage N-terminal to proline and between aspartic acid and proline (Trypsin/P + DP), and a maximum of two missed cleavages were allowed. Cysteine carbamidomethylation was set as fixed modification, and protein N-terminal acetylation, methionine oxidation, pyroglutamate for N-terminal glutamine, and phophorylation of serine, threonine, and tyrosine (phospho-STY) were selected as variable modifications. ...
Muscle "stem cells", or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential "waves" and underlined vast reprogramming in key processes governing cell fate decisions i.e. cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several "muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.
... AB-GADPH, Glyceraldehyde 3-phosphate dehydrogenase; GADPH2, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase isoform; ACLB1, ACLB2, ATP citrate lyase a-subunit; CS, Citrate synthase; ENO1, ENO2, ENO3, Enolase; FBA2, FBA3, Fructose-bisphosphate aldolase; FRK2, FRK3, Fructokinase; GPI1, GPI2, Glucose-6-phosphate isomerase; MDH, malate dehydrogenase; PEPC1, PEPC2, PEPC3 phosphoenolpyruvate carboxylase; PEPCK2, phosphoenolpyruvate carboxykinase; PGM, Phosphoglucomutase; PK, pyruvate kinase; TPI, Triosephosphate isomerase; Susy1, Sucrose synthase; TKL, Transketolase 1, 1,3BPG, 1,3-Bisphosphoglycerate; RuBP, Ribulose-1,5-bisphosphate; R5P, ribulose-5-phosphate; FDB Fructose-1,6-Bisphosphate; F6P fructose-1,6-bisphosphate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate, DHAP, dihydroxyacetone phosphate. 36 and Drosophila melanogaster 37 . In the latter case, mRNA and protein levels showed a fairly good Pearson correlation coefficient of r = 0.8, when genes not responding to the applied stimulus were removed from the analysis. ...
... However, the global-scale quantification of specific proteins is restricted due to the limited availability of antibody-based protein quantification methods. Bonaldi et al. 22 conducted a SILAC-based high throughput quantitative proteomic analysis following the silencing of a specific gene, paving the way for investigation of the global impact of RNAi on protein outcomes. Abdrakhmanova et al. 23 made a step forward by successfully combining RNAi with iTRAQ-based quantitative proteomics, which is a more accurate quantification method with relatively high sensitivity and reproducibility 8,24 . ...
Oxidative stress and apoptosis are involved in Ochratoxin A (OTA)-induced renal cytotoxicity. Apoptosis signal-regulating kinase 1 (ASK1) is a Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK, MAP3K) family member that plays an important role in oxidative stress-induced cell apoptosis. In this study, we performed RNA interference of ASK1 in HEK293 cells and employed an iTRAQ-based quantitative proteomics approach to globally investigate the regulatory mechanism of ASK1 in OTA-induced renal cytotoxicity. Our results showed that ASK1 knockdown alleviated OTA-induced ROS generation and Δψm loss and thus desensitized the cells to OTA-induced apoptosis. We identified 33 and 24 differentially expressed proteins upon OTA treatment in scrambled and ASK1 knockdown cells, respectively. Pathway classification and analysis revealed that ASK1 participated in OTA-induced inhibition of mRNA splicing, nucleotide metabolism, the cell cycle, DNA repair, and the activation of lipid metabolism. We concluded that ASK1 plays an essential role in promoting OTA-induced renal cytotoxicity.
... Instead, it may reflect a common mode of action for many factors that regulate transcription at the level of chromatin structure. To examine this possibility, we performed similar correlation analyses for transcriptional repression targets of two other chromatin effectors for which microarray data are available: from RNAi-dependent depletion of heterochromatin factor 1, HP1, in Drosophila Kc cells (Schwaiger et al. 2010) and RNAi knockdown of chromatin remodeler ISWI in Drosophila SL2 cells (Bonaldi et al. 2008). Both factors are likely to share regulatory roles with H1: HP1 and H1 physically interact and are known to participate in heterochromatin-related gene silencing (Lu et al. 2013), whereas ISWI was shown to be required for H1 deposition into chromatin (Corona et al. 2007;Siriaco et al. 2009). ...
Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and post-translational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity- and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression.
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... The effect on Kc cells appeared to be larger and as it was suspected that this would therefore result in larger changes at the proteome level we decided to continue all experiments with Kc cells (Suppl Figure 1; data for S2 cells not shown). Since no commercial SILAC medium is available for insect cell lines, medium depleted for arginine and lysine was prepared based on both the composition of commercially available Schneider's insect medium and protocols in the literature [43]. Details of the SILAC medium formula that was used in this study are given in Suppl Table 1. ...
The ecdysone signaling pathway plays a major role in various developmental transitions in insects. Recent advances in the understanding of ecdysone action have relied to a large extent on the application of molecular genetic tools in Drosophila. Here, we used a comprehensive quantitative SILAC mass spectrometry-based approach to study the global, dynamic proteome of a Drosophila cell line to investigate how hormonal signals are transduced into specific cellular responses. Global proteome data after ecdysone treatment after various time points were then integrated with transcriptome data. We observed a substantial overlap in terms of affected targets between the dynamic proteome and transcriptome, although there were some clear differences in timing effects. Also, downregulation of several specific mRNAs did not always correlate with downregulation of their corresponding protein counterparts and in some cases there was no correlation between transcriptome and proteome dynamics whatsoever. In addition, we performed a comprehensive interactome analysis of EcR, the major target of ecdysone. Proteins co-purified with EcR include factors involved in transcription, chromatin remodeling, ecdysone signaling, ecdysone biosynthesis and other signaling pathways. Novel ecdysone-responsive proteins identified in this study might link previously unknown proteins to the ecdysone signaling pathway and might be novel targets for developmental studies. To our knowledge, this is the first time that ecdysone signaling is studied by global quantitative proteomics. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... For choline deficiency and SILAC labeling, S2 cells were grown in custom Schneider medium (Bonaldi et al., 2008), with or without 1 mM choline, or 0.4 g/l 13 C 6 15 N 4 Larginine and 1.65 g/l 13 C 6 15 N 2 L-lysine (Sigma Isotec), respectively. (Gehrig et al., 2008). ...
... Drosophila S2 cells were grown in Schneider's medium supplemented with 10% (vol/vol) fetal calf serum at 26-28°C in 175-cm 2 tissue culture flasks. SILAC labeling was performed based on the method described in Bonaldi et al. (2008). Schneider's medium for SILAC labeling (without arginine and lysine) was purchased from Dundee Cell Products (Dundee, UK). ...
We have developed 'fractionation profiling', a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation, to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells, and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and report the first comprehensive insect clathrin-coated vesicle proteome. Importantly, the cluster analysis extends to all profiled proteins, and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterisation of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome, and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.
... Comparando-se a amostra submetida à interferência com uma amostra controle (sem interferência), as mudanças na expressão gênica ou em PTMs podem ser detectadas e quantificadas.Alguns trabalhos abrangendo estas combinações de metodologias tem sido publicados[47,74]. Em artigo publicado em 2008, Bonaldi e colaboradores[75] utilizaram RNAi para knockdown de uma subunidade de um complexo de remodelamento de cromatina (ISWI) em Drosophila melanogaster. O perfil proteico das células submetidas ao RNAi foi obtido por proteômica quantitativa (marcação por SILAC) e os resultados comparados com os dados de microarranjo, gerados por outro trabalho[76]. ...
Trypanosomatids have a complex life cycle switching between different hosts.
In this way, they find distinct environments where quick adaptation is needed. Among
the cell signaling mechanisms, protein phosphorylation, driven by protein kinases and
protein phosphatases, is an important process with potential to supply this necessity.
Some finds on Trypanosomatids’ genomes, such as, the absence of dedicated
tyrosine or tyrosine kinase-like receptors, the over-representation of some kinase
groups, the presence of unique kinases, and other, highlight the phosphorylation in
Trypanosomatids as a hot spot topic. The flagellate parasite Trypanosoma brucei
possesses 176 protein kinases classified in many families, among them, the mitogen
activated protein kinase (MAPKs) family. Several publications have been
documented the participation of this family in processes like cellular division and
differentiation, in response to stress conditions, and other. Nevertheless little is
known about its function in Trypanosomatids. In this sense, this work aimed to
evaluate the effects of MAPK depletion on T. brucei. As a start point, the participation
of MAPKs in the cellular proliferation was addressed. The results showed that almost
a half of MAPK knockdowns had the cell proliferation rate affected. One of these
knockdowns (Tb927.10.10870) was also quantitatively analyzed at the level of
proteins, phosphoproteins, and mRNA transcripts by mass spectrometry based
proteomics combined with metabolic labeling (SILAC) and by RNA-Seq. In total,
1,511 proteins, 1,510 phosphorylation sites, and nearly of 12 million of reads were
identified. Despite the cell proliferation rate was strongly affected in this knockdown,
few modulations in the proteome, phosphoproteome or transcriptome was observed.
In consonance with other works, this could represent the robustness of MAPK
signaling pathway in Trypanosomatids. Between the few modulations is worthy of
note some mitochondrial proteins and phosphorylation sites on proteins related to
mRNA stability control. As the MAPKs act in phosphorylation cascades, we also
evaluate the interaction between the members of this signaling pathway by two
hybrid assay. Among the 1,024 tested interactions, 22 were positive, showing no
overlap with the interactions described for their ortologues in model organisms. Take
together, the finds of this work have the potential to drive future projects in order to
well understand the MAPK signaling pathway in Trypanosomatids.
... 4,33 Therefore, quantitative proteomic analysis based on mass spectrometry represents an important approach to examine the cellular composition of proteins, independently of antibody or tracer technologies. 19,33,37 Because mass spectrometric instrumentation increased in sensitivity and sample preparation became more efficient in recent years, it is now feasible to perform proteomics with low amounts of zebrafish tissues even with larval hearts or single embryos. 12,38 To the best of our knowledge, our data sets provide the first quantitative proteome atlas of nine distinct adult zebrafish organs. ...
The zebrafish has become a widely used model organism employed for developmental studies, live cell imaging, and genetic screens. High-resolution transcriptional profiles of different development and adult stages and organs were generated and are readily accessible via the ZFIN database. In contrast, quantitative proteomic studies of zebrafish organs are still in its infancy. Here, we used the SILAC (stable isotope labeling by amino acids in cell culture) zebrafish as a "spike-in" reference to generate a protein atlas of nine organs including gills, brain, heart, muscle, liver, spleen, skin, swim bladder and testis. Single-shot 4h LC gradients coupled to a quadrupole-Orbitrap (QExactive) instrument allowed identification of over 5000 proteins in less than five days, of which more than 70% were quantified in triplicates. Identified proteins were subjected to BLAST searches and Gene Ontology classification to improve annotation of zebrafish proteins and obtain insights into potential functions. Comparison to mouse tissue proteome datasets revealed differences and similarities in the protein composition of zebrafish versus mouse organs. We reason that the dataset will be helpful for the proteomic characterization of zebrafish organs and identification of tissue-specific proteins that might serve as biomarkers. Our approach provides a complementary view into the biochemistry of zebrafish models and will assist large-scale protein quantification in zebrafish disease models.
... A broader utility of MS-based proteomics as an indispensable part of systems biology approaches is evident from emerging integrative studies that combine proteomics with other high-throughput methods, such as transcriptomics. In one example, a chromatin remodeling regulator ISWI was knocked down by RNAi in Drosophila SL2 cells, and the resulting global changes in the transcriptome and the proteome were quantified 64 . This study revealed a widespread non-correlation between changes in protein and transcript levels, suggesting a significant extent of post-transcriptional control of protein abundance in response to perturbation of a major chromatin component. ...
Mass spectrometry (MS)‐based proteomics has become an indispensable tool for protein identification and quantification. In this paper, common MS workflows are described, with an emphasis on applications of MS‐based proteomics in developmental biology. Progress has been made in the analysis of proteome changes during tissue differentiation and in various genetic perturbations. MS‐based proteomics has been particularly useful for identifying novel protein interactions by affinity purification‐mass spectrometry (AP‐MS), many of which have been subsequently functionally validated and led to the discovery of previously unknown modes of developmental regulation. Quantitative proteomics approaches can be used to study posttranslational modifications (PTMs) of proteins such as phosphorylation, to reveal the dynamics of intracellular signal transduction. Integrative approaches combine quantitative MS‐based proteomics with other high‐throughput methods, with the promise of a systems level understanding of developmental regulation. WIREs Dev Biol 2013, 2:723–734. doi: 10.1002/wdev.102
This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes
Technologies > Analysis of Proteins
... It has been shown that senescent cells display a distinct gene expression profile at the transcriptome level [15], with such "omic" studies mostly focused on replicative senescence [16,17]. Moreover, transcriptome dynamics do not reflect direct changes at the proteome level [18,19]. Hence, a mechanistic investigation of the proteome in oncogene induced senescent cells could provide information about differential regulation of responsive genes in the senescence program. ...
Cellular senescence, an irreversible cell cycle arrest induced by a diversity of stimuli, has been considered as an innate tumor suppressing mechanism with implications and applications in cancer therapy. Using a targeted proteomics approach we show that fibroblasts induced into senescence by expression of oncogenic Ras exhibit a decrease of global acetylation on all core histones, consistent with formation of senescence-associated heterochromatic foci. We also detected clear increases in repressive markers (e.g., >50% elevation of H3K27me2/3) along with decreases in histone marks associated with increased transcriptional expression/elongation (e.g., H3K36me2/3). Despite the increases in repressive marks of chromatin, 179 loci (of 2206 total) were found to be upregulated by global quantitative proteomics. The changes in the cytosolic proteome indicated an upregulation of mitochondrial proteins and downregulation of proteins involved in glycolysis. These alterations in primary metabolism are opposite of the well-known Warburg effect observed in cancer cells. This study significantly improves our understanding of stress-induced senescence and provides a potential application for triggering it in anti-proliferative strategies that target the primary metabolism in cancer cells. This article is protected by copyright. All rights reserved.
... LD Purification-For LD purification, ten 75-cm 2 cell culture dishes of Drospohila S2 cells were used. Stable isotope labeling of amino acids in cell culture (SILAC) of the S2 cells was performed as previously described, and the labeling efficiency was Ͼ98% before the last passage (35). LD formation was induced by incubating confluent cells with 1 mM oleate complexed to BSA (36) overnight. ...
Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase, surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum (ER), mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, confident identification of bona fide LD proteins by proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles (PCP). Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profile to that of known LD proteins. Application of the PCP strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction where 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins by microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins are both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. Moreover, our results enable the fast detection of changes of the LD proteome due to depletion of potential LD targeting factors, such as Arf79F.
... In the hypothetical case of pure transcriptional regulation the correlation between these two levels would be near one, and would only be limited by the technical limitations and imperfections of the respective quantitative profiling technologies. Indeed, while early investigations found low or no correlation between proteome and transcriptome[6,7], recently much higher levels of global correlation have been reported[8,9]. While transcriptional regulation is generally a dominant aspect of the entire expression cascade, there are many known examples of posttranscriptional regulation like micro-RNA controlled inhibition of transcripts[10]and directed protein degradation[11]. ...
Quantitative proteomics now provides abundance ratios for thousands of proteins upon perturbations. These need to be functionally interpreted and correlated to other types of quantitative genome-wide data such as the corresponding transcriptome changes. We describe a new method, 2D annotation enrichment, which compares quantitative data from any two 'omics' types in the context of categorical annotation of the proteins or genes. Suitable genome-wide categories are membership of proteins in biochemical pathways, their annotation with gene ontology terms, sub-cellular localization, presence of protein domains or membership in protein complexes. 2D annotation enrichment detects annotation terms whose members show consistent behavior in one or both of the data dimensions. This consistent behavior can be a correlation between the two data types, such as simultaneous up- or down-regulation in both data dimensions, or a lack thereof, such as regulation in one dimension but no change in the other. For the statistical formulation of the test we introduce a two-dimensional generalization of the nonparametric two-sample test. The false discovery rate is stringently controlled by correcting for multiple hypothesis testing. We also describe one-dimensional annotation enrichment, which can be applied to single omics data. The 1D and 2D annotation enrichment algorithms are freely available as part of the Perseus software.
Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light‐induced damage, or dietary deficiencies. Large‐scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC‐MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.
Omics, including genomics, proteomics, and metabolomics, enable us to explain symbioses in terms of the underlying molecules and their interactions. The central task is to transform molecular catalogs of genes, metabolites, etc., into a dynamic understanding of symbiosis function. We review four exemplars of omics studies that achieve this goal, through defined biological questions relating to metabolic integration and regulation of animal-microbial symbioses, the genetic autonomy of bacterial symbionts, and symbiotic protection of animal hosts from pathogens. As omic datasets become increasingly complex, computationally sophisticated downstream analyses are essential to reveal interactions not evident from visual inspection of the data. We discuss two approaches, phylogenomics and transcriptional clustering, that can divide the primary output of omics studies—long lists of factors—into manageable subsets, and we describe how they have been applied to analyze large datasets and generate testable hypotheses.
Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the United States and Europe (Dolk et al., 2010; Heron et al., 2009). Of the congenital malformations, congenital heart disease (CHD) is the most common (Dolk et al., 2010; Heron et al., 2009). Thus, a molecular understanding of heart development is an essential goal for improving clinical approaches to CHD. However, CHDs are commonly a result of genetic defects that manifest themselves in a spatial and temporal manner during the early stages of embryogenesis, leaving them mostly intractable to mass spectrometry-based analysis. Here, we describe the technologies and advancements in the field of mass spectrometry over the past few years that have begun to provide insights into the molecular and cellular basis of CHD and prospects for these types of approaches in the future.
Successful cell culture depends on successful formulation of appropriate cell culture media and growth conditions. Defining the "milieu interieur" of Drosophila hemolymph provided a starting point for defining appropriate osmolarity, pH, ionic content, amino acids, and sugars, and so on to support Drosophila cell culture. Supplementation with sources of vitamins, such as from yeast extract, serum, and hormones or growth factors further influence growth of cells in culture. This work has resulted in definition of a number of standard media recipes that are presented and discussed in the chapter along with background information.
Perhaps more than any other organism, the fruit fly Drosophila melanogaster has been the vanguard for molecular genetics and genome mapping. One of the first few metazoan genomes to be sequenced, the fly also offers a set of unparalleled molecular and genetic tools for exploring gene function and genome organization in a complex multicellular animal. In this chapter, we give a brief history of a century of genome biology with the fly, starting with the work in the famous fly room at Columbia University and leading to genome projects and multinational collaborations characteristic of current biological research. We discuss recent technical developments for the analysis of the genome, the proteome, and the interactome of D. melanogaster and related species, finishing with insights emerging from the recent modENCODE project aimed at cataloging all functional elements in the fly genome. Each section concludes with the things to come-“The Times They Are a-Changin.'" May You Live in Interesting Times Astounding Science Fiction Magazine 1950 The Times They Are a-Changin’ Bob Dylan 1963.
Diapause is a physiological adaptation that allows an organism to survive adverse environmental conditions. Diapause occurs at a specific developmental stage in each species. There are few reports regarding the molecular regulatory mechanism of nymphal diapause in Laodelphax striatellus, which is an important graminaceous crop pest. Our previous studies identified the conditions for nymphal diapause in this species. Here, we combined RNA sequencing transcriptomics and quantitative proteomic analyses to identify nymphal diapause-related genes and proteins. The analysis of differentially regulated genes identified four gene/protein pairs that were synchronously up-regulated, and six gene/protein pairs that were synchronously down-regulated, suggesting that these genes may regulate nymphal diapause. The up-regulated gene juvenile hormone acid methyl transferase (JHAMT) and the down-regulated gene cytochrome P450 monooxygenase (CYP314A1, Shd) were chosen for further functional studies. After knocking-down of LsJHAMT and LsShd in vivo by RNA interference, the titer of JH III and 20E decreased significantly, and the duration of the nymphal development period was severely altered. Thus LsJHAMT and LsShd regulated JH III and 20E titers in the hemolymph to control the nymphal diapause status. This study may lead to new information on the regulation nymphal diapause of this important agricultural insect pest.
Chitin synthases are very important enzymes for chitin synthesis in various species, which makes them a specific target of insecticides. In the present study, we investigated the function of the chitin synthase A (CHSA) gene isolated from Mythimna separata. The majority of dsMysCHSA treated larvae (89.50%) exhibited lethal phenotypes, including three phenotypes with severe cuticle deformations. The dsMysCHSA treatment in adult females affected oogenesis, and significantly reduced the ovary size and the oviposition number compared with controls. To determine how MysCHSA affected female fecundity, we performed combined analyses of RNA-sequencing (RNA-Seq) transcriptome and TMT proteome (tandem mass tags) data in M. separata after treatment with MysCHSA-RNAi. The differentially expressed proteins and genes affected fecundity-related proteins, energy metabolism, fatty acid metabolism, amino sugars and nucleotide sugar metabolism pathways. Taken together, these results suggested that MysCHSA acts on M. separata ecdysis and fecundity, and has the potential as a target gene for pest control.
Significance:
The field of cell signaling is a promising topic of study for trypanosomatids, since little is known about this topic and the gene expression regulation occurs at post-transcriptional level. In this sense, the present work increases the knowledge on protein phosphorylation process in Trypanosoma brucei. We depleted one MAP kinase (MAPKLK1) of T. brucei and evaluated the effects on the cell. We showed that MAPKLK1 is essential to the cell, while few modulations on phosphoproteome, proteome and transcriptome are observed with its depletion. Although in low number, the changes in phosphoproteome were significant, presenting possible substrate candidates of MAPKLK1 and indirect targets related to mRNA processing and stability control, metabolic pathways, among others. This result provides insights in the phosphorylation network of T. brucei, a model organism that impacts human and animal health.
Recently, transcriptome and proteome data have increasingly been used to identify potential novel genes related to insect phenotypes. However, there are few studies reporting the large-scale functional identification of such genes in insects. To identify novel genes related to fecundity in the brown planthopper (BPH), Nilaparvata lugens, 115 genes were selected from the transcriptomic and proteomic data previously obtained from high- and low-fecundity populations in our laboratory. The results of RNA interference (RNAi) feeding experiments showed that 91.21% of the genes were involved in the regulation of vitellogenin (Vg) expression and may influence BPH fecundity. After RNAi injection experiments, 12 annotated genes were confirmed as fecundity-related genes and three novel genes were identified in the BPH. Finally, C-terminal binding protein (CtBP) was shown to play an important role in BPH fecundity. Knockdown of CtBP not only led to lower survival, underdeveloped ovaries and fewer eggs laid but also resulted in a reduction in Vg protein expression. The novel gene resources gained from this study will be useful for constructing a Vg regulation network and may provide potential target genes for RNAi-based pest control.
Not all genes are equally important during the process of speciation. This premise underlies a basic question in evolutionary biology-"Does divergence at any one gene, or set of genes, play a particularly important role in speciation?" The answer to this question may appear to be no for some forms of reproductive isolation, but there may indeed be 'kinds of genes' that routinely play a role in speciation when divergence is driven by postmating, prezygotic traits. Here, I outline the kinds of molecular pathways and interactions that underlie postmating, prezygotic phenotypes which ultimately points to the kinds of genes where we should look for species-specificity. Interestingly, it is only when we consider the entire system of interacting sex proteomes, cell structure, membrane dynamics, and physiological pathways that a picture of where species-specific interactions likely occur becomes clear. While this approach points to several kinds of pathways and gene types, a notable finding is that cell membrane receptors (like G-protein coupled receptors and receptor tyrosine kinases) that line the inside of the female reproductive tract and trigger postcopulation cell signaling should be considered the kinds of genes that routinely contribute to reproductive isolation and speciation when divergence is driven by postmating, prezygotic phenotypes.
This chapter discusses the insect genome sequencing and analysis of sequenced genomes using “omics” and high-throughput sequencing technologies. It also provides an overview of proteomics and structural genomics of insect systems biology. Research on insects, especially in the areas of physiology, biochemistry, and molecular biology, has undergone notable transformations during the last two decades. Almost all insect genomes sequenced to date employed the whole-genome shotgun sequencing (WGS) method. Shotgun genome sequencing begins with isolation of high molecular weight genomic DNA from nuclei isolated from isogenic lines of insects. The genomic DNA is then randomly sheared, end-polished with Bal31 nuclease/ T4 DNA polymerase primers, and, finally, the DNA is size-selected. The size-selected, sheared DNA is then ligated to restriction enzyme adaptors such as the BstX1 adaptors. Subsequently, the genomic fragments are inserted into restriction enzyme-linearized plasmid vectors. Most genomes sequenced to date employed this technology. Sanger sequencing must be distinguished from next generation sequencing technology, which has entered the marketplace during the last four years and is rapidly changing the approaches used to sequence genomes. Genomes sequenced by NGS technologies will be completed more quickly and at a lower price than those from the first few insect genomes.
The fruit fly Drosophila melanogaster is one of the most widely used and well-studied model organisms in biology and therefore a promising tool for quantitative proteomics. Here, we describe a method to label D. melanogaster with stable isotope labeled amino acids in vivo. Feeding flies with heavy lysine labeled yeast cells leads to virtually complete heavy labeling already in the first filial generation. The approach is simple, fast, and cost-effective, which makes SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.Key words
Drosophila melanogaster
SILACIn vivo labelingYeastProtein extraction
Stable isotope labeling by amino acids in cell culture (SILAC) is an established and potent method for quantitative proteomics. When combined with high-resolution mass spectrometry (MS) and efficient algorithms for the analysis of quantitative MS data, SILAC has proven to be the strategy of choice for the in-depth characterization of functional states at the protein level. The fruit fly Drosophila melanogaster is one of the most widely used model systems for studies of genetics and developmental biology. Despite this, a global proteomic approach in Drosophila is rarely considered. Here, we describe an adaptation of SILAC for functional investigation of fruit flies by proteomics: We illustrate how to perform efficient SILAC labeling of cells in culture and whole fly larvae. The combination of SILAC, a highly accurate global protein quantification method, and of the fruit fly, the prime genetics and developmental model, represents a unique opportunity for quantitative proteomic studies in vivo.
The large-scale identification and quantification of proteins is an important foundation of systems biology. Here we focus on the particularly powerful technology of mass spectrometry (MS)-based proteomics, with an emphasis on recent high-resolution and quantitative approaches. MS-based proteomics is used to characterize proteins in complex mixtures and it is now possible to quantify nearly all the proteins in human cell lines. Subcellular localization and protein turnover can also be addressed comprehensively. In affinity purifications, quantitative proteomics distinguishes specific interacting proteins from background binders. Thousands of phosphorylation sites as well as other post-translational modifications can readily be quantified in vivo, providing direct access to cellular information processing events. Underlying the recent success of the field are developments in computational proteomics, which now allow highly sophisticated and completely automatic analysis of raw MS data and streamlined bioinformatic and systems-level interpretation of the results.
Acoustic signals produced to attract mates before, during, and after courtship are frequently involved with sexual selection, sexual isolation, and reproductive isolation in Drosophila spp. and other animals, yet few studies have revealed how courtship songs evolve in a larger phylogenetic context. Therefore, we mapped different acoustic components of courtship songs in the monophyletic Drosophila buzzatii species cluster onto an independently derived period (per) gene + chromosome inversion phylogeny to assess the concordance of courtship song evolution with species divergence. These cactophilic flies are distributed throughout several biomes in southern South America and include the sibling species D. buzzatii, D. koepferae, D. serido, D. borborema, D. seriema, D. antonietae, and D. gouveai. All seven species produced two song types; primary and secondary pulse songs, except for D. borborema and D. gouveai that produced no secondary songs. Courtship songs were characterized by analyzing six commonly studied acoustic components including burst duration (BD), carrier frequency (CF), pulse length (PL), pulse number (PN), inter-burst interval (IBI), and inter-pulse interval (IPI). Significant intra-and inter-specific song variation was observed for BD, PN, and IBI, while CF, PL, and IPI varied in a more species-specific manner, albeit with some overlap. Thus, some song components may be better species recognition signals than others. Multivariate clustering analyses resolved all species into distinct, nonoverlapping groups. Mapping individual song traits (BD, IBI, and IPI) as well composites of these song variables onto our (per) gene + chromosome inversion phylogeny revealed no phylogenetic signal when different comparative mapping methods were used. Hence, the evolution of courtship songs in D. buzzatii cluster species was uncorrelated with the degree of species divergence. These findings reinforce previous observations that courtship songs evolve rapidly enough to erase any signature of evolutionary affinity between closely related animal species.
Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC) protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA), which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.
Drosophila melanogaster is one of the most widely used model organisms in life sciences. Mapping its proteome is of great significance for understanding the biological characteristics and tissue functions of this species. However, the comprehensive coverage of its proteome remains a challenge. Here we describe a high-coverage analysis of whole fly through a one-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. By combining the datasets of two types of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two kinds of tagmata, the high-coverage analysis resulted in the identification of 5,262 genes, which correspond to 38.23% of the entire coding genes. Moreover, we found that the fly head and body have different molecular weight distributions of their proteomes when the proteins were resolved with SDS-PAGE and image analysis of the stained gel. This phenomenon was further confirmed by both label-free and isobaric tags for relative and absolute quantitation-based quantitative approaches. The consistent results of the two different quantitation methods also demonstrated the stability and accuracy of the LC-MS/MS platform. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifiers PXD000454 and PXD000455.
Small regulatory RNAs (microRNAs, siRNAs, and piRNAs) exhibit several unique features that clearly distinguish them from other known gene regulators. Their genomic organization, mode of action, and proposed biological functions raise specific questions. In this review, we focus on the quantitative aspect of small regulatory RNA biology. The original nature of these small RNAs accelerated the development of novel detection techniques and improved statistical methods and promoted new concepts that may unexpectedly generalize to other gene regulators. Quantification of natural phenomena is at the core of scientific practice, and the unique challenges raised by small regulatory RNAs have prompted many creative innovations by the scientific community.
Microtubules dramatically change their dynamics and organisation at entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we use an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins in which most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localisation. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that suppressing microtubule affinity of this mitotic MAP in combination with nuclear localisation is essential for microtubule organisation in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.
Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced in vivo into select multi-cellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled Drosophila melanogaster can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the using heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. In vivo conversion of heavy lysine and/or heavy arginine to several non-essential amino acids was observed in labeled fly, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence dependent to be more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval-pupa transition while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions.
Omics, including genomics, proteomics, and metabolomics, enable us to explain symbioses in terms of the underlying molecules and their interactions. The central task is to transform molecular catalogs of genes, metabolites, etc., into a dynamic understanding of symbiosis function. We review four exemplars of omics studies that achieve this goal, through defined biological questions relating to metabolic integration and regulation of animal-microbial symbioses, the genetic autonomy of bacterial symbionts, and symbiotic protection of animal hosts from pathogens. As omic datasets become increasingly complex, computationally sophisticated downstream analyses are essential to reveal interactions not evident from visual inspection of the data. We discuss two approaches, phylogenomics and transcriptional clustering, that can divide the primary output of omics studies-long lists of factors-into manageable subsets, and we describe how they have been applied to analyze large datasets and generate testable hypotheses.
Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.
Sequence analysis of the human genome and the association of genetic aberrations with diseases have provided a rough framework whereby the impact of individual genotypes can be assessed. To fully understand the effect of individual and co-occurring genetic aberrations, as well as their individual and collected contribution to the development of diseases, it is critical to analyse the matching proteome and to determine how the organisation, expression level and function of protein networks are affected. Sensitive mass spectrometric platforms in combination with innovative workflows allow qualitative and quantitative analyses of the cellular as well as the extracellular proteome. Importantly, in addition to specifically identifying the content of the proteome, several aspects of the proteomic organisation can be analysed including protein complexes, protein modifications, enzymatic activities and subcellular/organelle localisation. Together, these measurements will provide novel insight into the biological effect of disease-causing mutations ultimately coupling genotype and phenotype.
The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is approximately 3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.
NURF is an ISWI complex of four proteins that uses the energy of ATP hydrolysis to catalyze nucleosome sliding. Three NURF components have been identified previously. We have cloned cDNA encoding the largest NURF subunit, revealing a 301 kDa polypeptide (NURF301) that shares structural motifs with ACF1. We have reconstituted full and partial NURF complexes from recombinant proteins and show that NURF301 and the ISWI ATPase are necessary and sufficient for accurate and efficient nucleosome sliding. An HMGA/HMGI(Y)-like domain of NURF301 that facilitates nucleosome sliding indicates the importance of DNA conformational changes in the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, providing a basis for targeted recruitment of the NURF complex to specific genes.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA-target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.
The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.
The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.
The ATPase ISWI is the molecular motor of several remodeling factors that trigger nucleosome sliding in vitro. In search for the underlying mechanism, we found that unilateral binding of ISWI to a model nucleosome correlated with directional movement of the nucleosome toward the enzyme. It has been proposed that ISWI might loosen histone-DNA interactions through twisting DNA. However, nucleosome sliding assays on nicked DNA substrates suggest that propagation of altered twist is not involved. Surprisingly, nicks in the linker DNA in front of the nucleosome facilitate sliding. These data suggest that the rate of nucleosome sliding is limited by a conformational change other than twisting, such as the formation of a short loop, of DNA at the entry into the nucleosome.
RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, we isolated mutations in the gene encoding the large NURF subunit, nurf301. We confirm that NURF is required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, we show that NURF is required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form.
Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.
Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in "active" (e.g. phosphorylated) and "control" (e.g. non-phosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, B., Kratchmarova, I., Ong, S.-E., Nielsen, M., Foster, L. J., and Mann, M. (2003) Nat. Biotechnol. 21, 315-318). A tyrosine-phosphorylated peptide of the epidermal growth factor receptor specifically retrieved the Src homology domain (SH) 2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins as specific binding partners. Two of these, Pacsin 3 and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline-SH3 domain switch.
MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.
Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. Here we investigate the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus.
Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs.
To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.
Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.
RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of approximately 21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, 'off-target effects' compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.
Off-target effects are one of the most serious problems in RNA interference (RNAi). Here, we present dsCheck (http://dsCheck.RNAi.jp/), web-based online software for estimating off-target effects caused by the long double-stranded RNA (dsRNA) used in RNAi
studies. In the biochemical process of RNAi, the long dsRNA is cleaved by Dicer into short-interfering RNA (siRNA) cocktails.
The software simulates this process and investigates individual 19 nt substrings of the long dsRNA. Subsequently, the software
promptly enumerates a list of potential off-target gene candidates based on the order of off-target effects using its novel
algorithm, which significantly improves both the efficiency and the sensitivity of the homology search. The website not only
provides a rigorous off-target search to verify previously designed dsRNA sequences but also presents ‘off-target minimized’
dsRNA design, which is essential for reliable experiments in RNAi-based functional genomics.
Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.
Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, we performed whole genome expression analysis on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling.
RNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21 000 double-stranded RNAs targeting known and predicted genes
in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated
from these screens are collected in the DRSC database (http://flyRNAi.org/cgi-bin/RNAi_screens.pl) in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is
to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows
detailed examination of the data and provides access to a number of other databases and bioinformatics tools.
The field of proteomics is built on technologies to analyze large numbers of proteins—ideally the entire proteome—in the same experiment. Mass spectrometry (MS) has been successfully used to characterize proteins in complex mixtures, but results so far have largely been qualitative. Two recently developed methodologies offer the opportunity to obtain quantitative proteomic information. Comparing the signals from the same peptide under different conditions yields a rough estimate of relative protein abundance between two proteomes. Alternatively, and more accurately, peptides are labeled with stable isotopes, introducing a predictable mass difference between peptides from two experimental conditions. Stable isotope labels can be incorporated 'post-harvest', by chemical approaches or in live cells through metabolic incorporation. This isotopic handle facilitates direct quantification from the mass spectra. Using these quantitative approaches, precise functional information as well as temporal changes in the proteome can be captured by MS.
A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.
Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage.
To probe the yeast proteome in depth and determine factors currently preventing complete analysis, we grew yeast cells, extracted proteins and separated them by one-dimensional gel electrophoresis. Peptides resulting from trypsin digestion were analyzed by liquid chromatography mass spectrometry on a linear ion trap-Fourier transform mass spectrometer with very high mass accuracy and sequencing speed. We achieved unambiguous identification of more than 2,000 proteins, including very low abundant ones. Effective dynamic range was limited to about 1,000 and effective sensitivity to about 500 femtomoles, far from the subfemtomole sensitivity possible with single proteins. We used SILAC (stable isotope labeling by amino acids in cell culture) to generate one-to-one pairs of true peptide signals and investigated if sensitivity, sequencing speed or dynamic range were limiting the analysis.
Advanced mass spectrometry methods can unambiguously identify more than 2,000 proteins in a single proteome. Complex mixture analysis is not limited by sensitivity but by a combination of dynamic range (high abundance peptides preventing sequencing of low abundance ones) and by effective sequencing speed. Substantially increased coverage of the yeast proteome appears feasible with further development in software and instrumentation.
RNA interference (RNAi) screening is a powerful technology for functional characterization of biological pathways. Interpretation of RNAi screens requires computational and statistical analysis techniques. We describe a method that integrates all steps to generate a scored phenotype list from raw data. It is implemented in an open-source Bioconductor/R package, cellHTS (http://www.dkfz.de/signaling/cellHTS). The method is useful for the analysis and documentation of individual RNAi screens. Moreover, it is a prerequisite for the integration of multiple experiments.
miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell
culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins
was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that
have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3′-untranslated
region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used
for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins.
Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.
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In addition to delivering a haploid genome to the egg, sperm have additional critical functions, including egg activation, origination of the zygote centrosome and delivery of paternal factors. Despite this, existing knowledge of the molecular basis of sperm form and function is limited. We used whole-sperm mass spectrometry to identify 381 proteins of the Drosophila melanogaster sperm proteome (DmSP). This approach identified mitochondrial, metabolic and cytoskeletal proteins, in addition to several new functional categories. We also observed nonrandom genomic clustering of sperm genes and underrepresentation on the X chromosome. Identification of widespread functional constraint on the proteome indicates that sexual selection has had a limited role in the overall evolution of D. melanogaster sperm. The relevance of the DmSP to the study of mammalian sperm function and fertilization mechanisms is demonstrated by the identification of substantial homology between the DmSP and proteins of the mouse axoneme accessory structure.
We report a method for large-scale absolute protein expression measurements (APEX) and apply it to estimate the relative contributions of transcriptional- and translational-level gene regulation in the yeast and Escherichia coli proteomes. APEX relies upon correcting each protein's mass spectrometry sampling depth (observed peptide count) by learned probabilities for identifying the peptides. APEX abundances agree with measurements from controls, western blotting, flow cytometry and two-dimensional gels, as well as known correlations with mRNA abundances and codon bias, providing absolute protein concentrations across approximately three to four orders of magnitude. Using APEX, we demonstrate that 73% of the variance in yeast protein abundance (47% in E. coli) is explained by mRNA abundance, with the number of proteins per mRNA log-normally distributed about approximately 5,600 ( approximately 540 in E. coli) protein molecules/mRNA. Therefore, levels of both eukaryotic and prokaryotic proteins are set per mRNA molecule and independently of overall protein concentration, with >70% of yeast gene expression regulation occurring through mRNA-directed mechanisms.
Drosophila melanogaster exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome.
Current methods for phosphoproteome analysis have several limitations. First, most methods for phosphopeptide enrichment lack the specificity to truly purify phosphopeptides. Second, fragmentation spectra of phosphopeptides, in particular those of phosphoserine and phosphothreonine containing peptides, are often dominated by the loss of the phosphate group(s) and therefore lack the information required to identify the peptide sequence and the site of phosphorylation, and third, sequence database search engines and statistical models for data validation are not optimized for the specific fragmentation properties of phosphorylated peptides. Consequently, phosphoproteomic data are characterized by large and unknown rates of false positive and false negative phosphorylation sites. Here we present an integrated chemical, mass spectrometric and computational strategy to improve the efficiency, specificity and confidence in the identification of phosphopeptides and their site(s) of phosphorylation. Phosphopeptides were isolated with high specificity through a simple derivatization procedure based on phosphoramidate chemistry. Identification of phosphopeptides, their site(s) of phosphorylation and the corresponding phosphoproteins was achieved by the optimization of the mass spectrometric data acquisition procedure, the computational tools for database searching and the data post processing. The strategy was applied to the mapping of phosphorylation sites of a purified transcription factor, dFOXO and for the global analysis of protein phosphorylation of Drosophila melanogaster Kc167 cells.
Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.
The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50-75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.
Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.
ATP-dependent chromatin-remodeling machines of the SWI/SNF family are involved in many cellular processes in eukaryotic nuclei, such as transcription, replication, repair and recombination. Remodeling factors driven by the ATPase ISWI make up a subgroup of this family that exhibits defined mechanistic and functional characteristics. ISWI-induced nucleosome mobility endows nucleosomal arrays with dynamic properties and recent results suggest that ISWI-type remodelers have diverse functions that range from transcriptional regulation to chromatin assembly and maintenance of chromosome structure.
Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
Proteomic analysis of complex protein mixtures using proteolytic digestion and liquid chromatography in combination with tandem mass spectrometry is a standard approach in biological studies. Data-dependent acquisition is used to automatically acquire tandem mass spectra of peptides eluting into the mass spectrometer. In more complicated mixtures, for example, whole cell lysates, data-dependent acquisition incompletely samples among the peptide ions present rather than acquiring tandem mass spectra for all ions available. We analyzed the sampling process and developed a statistical model to accurately predict the level of sampling expected for mixtures of a specific complexity. The model also predicts how many analyses are required for saturated sampling of a complex protein mixture. For a yeast-soluble cell lysate 10 analyses are required to reach a 95% saturation level on protein identifications based on our model. The statistical model also suggests a relationship between the level of sampling observed for a protein and the relative abundance of the protein in the mixture. We demonstrate a linear dynamic range over 2 orders of magnitude by using the number of spectra (spectral sampling) acquired for each protein.
With the sequencing of the human genome and the genomes of most major model organisms completed, the systematic characterisation
of gene functions remains a key challenge. During the past few years, RNA interference (RNAi) has become a powerful tool to
silence the expression of genes and analyse their loss-of-function phenotype when mutant alleles are not available. Genome-wide
RNAi screens against all predicted genes have been successfully used to dissect a variety of biological processes in Caenorhabditis elegans. Recently, a genome-wide library of double-stranded RNAs, that target every gene in the Drosophila genome and that is suitable for high throughput cell-based assays, was published. In this paper, recent advances will be
summarised. Screening strategies and applications as a route to comprehensively characterising gene function will be discussed.
During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.
This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.
RNA interference (RNAi) has become a powerful genetic approach to systematically dissect gene function on a genome-wide scale.
Owing to the penetrance and efficiency of RNAi in invertebrates, model organisms such as Drosophila melanogaster and Caenorhabditis elegans have contributed significantly to the identification of novel components of diverse biological pathways, ranging from early
development to fat storage and aging. For the correct assessment of phenotypes, a key issue remains the stringent quality
control of long double-stranded RNAs (dsRNA) to calculate potential off-target effects that may obscure the phenotypic data.
We here describe a web-based tool to evaluate and design optimized dsRNA constructs. Moreover, the application also gives
access to published predesigned dsRNAs. The E-RNAi web application is available at http://e-rnai.dkfz.de/.
Gene expression is regulated at multiple levels, and cells need to integrate and coordinate different layers of control to implement the information in the genome. Post-transcriptional levels of regulation such as transcript turnover and translational control are an integral part of gene expression and might rival the sophistication and importance of transcriptional control. Microarray-based methods are increasingly used to study not only transcription but also global patterns of transcript decay and translation rates in addition to comprehensively identify targets of RNA-binding proteins. Such large-scale analyses have recently provided supplementary and unique insights into gene expression programs. Integration of several different datasets will ultimately lead to a system-wide understanding of the varied and complex mechanisms for gene expression control.
A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.
Historically, chromatin has been subdivided into heterochromatin, transcriptionally inactive regions that remain densely packaged throughout the cell cycle, and euchromatin, transcriptionally active regions that take on a diffuse appearance as the cell enters interphase. The banded portion of the small fourth chromosome (dot chromosome) of Drosophila melanogaster is unusual in exhibiting many characteristics of heterochromatic domains, and at the same time maintaining a gene density typical of euchromatin. Similar to genes embedded in pericentric heterochromatin, many of the dot chromosome genes have adapted to a heterochromatic environment. Little is known about the regulation of these genes and less about their evolution in a chromatin context. Interestingly, most of the genes from the D. melanogaster fourth chromosome remain clustered on a small chromosome throughout the genus Drosophila; yet the dot chromosome appears euchromatic in some species, such as D. virilis. Existing genomic sequence data allow an exploration of the underlying differences in DNA sequence organization between species. Here we review the available data describing the dot chromosome, which derives primarily from D. melanogaster. With its unusual and changing nature, the dot chromosome in the genus Drosophila provides a unique opportunity for the examination of transitions between chromatin states during evolution.
Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.
Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster that binds to heterochromatin protein 1 (HP1) and has been implicated in heterochromatin-induced gene silencing. Heretofore, HP1 has been the only known binding partner of HP2, a large protein devoid of sequence motifs other than a pair of AT hooks. In an effort to identify proteins that interact with HP2 and assign functions to its various domains, nuclear proteins were fractionated under nondenaturing conditions. On separation of nuclear proteins, nucleosome assembly protein 1 (Nap-1) has an overlapping elution profile with HP2 (assayed by Western blot) and has been identified by mass spectrometry in fractions with HP2. Upon probing fractions in which HP2 and Nap-1 are both present, we find that the nucleosome remodeling factor (NURF), an ISWI-dependent chromatin remodeling complex, is also present. Results from coimmunoprecipitation experiments suggest that HP2 interacts with Nap-1 as well as with NURF; NURF appears to interact directly with both HP2 and Nap-1. Three distinct domains within HP2 mediate the interaction with NURF, allowing us to assign NURF binding domains in addition to the AT hooks and HP1 binding domains already mapped in HP2. Mutations in Nap-1 are shown to suppress position effect variegation, suggesting that Nap-1 functions to help to assemble chromatin into a closed form, as does HP2. On the basis of these interactions, we speculate that HP2 may cooperate with these factors in the remodeling of chromatin for silencing.