Natural Genetic Variation Differentially Affects the Proteome and Transcriptome in Caenorhabditis elegans
2016; Elsevier BV; Volume: 15; Issue: 5 Linguagem: Inglês
10.1074/mcp.m115.052548
ISSN1535-9484
AutoresPolina Kamkina, Basten L. Snoek, Jonas Grossmann, Rita Volkers, Mark G. Sterken, Michael Daube, Bernd Roschitzki, Claudia Fortes, Ralph Schlapbach, Alexander Röth, Christian von Mering, Michael O. Hengartner, Sabine Schrimpf, Jan E. Kammenga,
Tópico(s)CRISPR and Genetic Engineering
ResumoNatural genetic variation is the raw material of evolution and influences disease development and progression. An important question is how this genetic variation translates into variation in protein abundance. To analyze the effects of the genetic background on gene and protein expression in the nematode Caenorhabditis elegans, we quantitatively compared the two genetically highly divergent wild-type strains N2 and CB4856. Gene expression was analyzed by microarray assays, and proteins were quantified using stable isotope labeling by amino acids in cell culture. Among all transcribed genes, we found 1,532 genes to be differentially transcribed between the two wild types. Of the total 3,238 quantified proteins, 129 proteins were significantly differentially expressed between N2 and CB4856. The differentially expressed proteins were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlates more strongly than protein abundance versus transcript abundance within each wild type. Our findings indicate that in C. elegans only a fraction of the changes in protein abundance can be explained by the changes in mRNA abundance. These findings corroborate with the observations made across species. Natural genetic variation is the raw material of evolution and influences disease development and progression. An important question is how this genetic variation translates into variation in protein abundance. To analyze the effects of the genetic background on gene and protein expression in the nematode Caenorhabditis elegans, we quantitatively compared the two genetically highly divergent wild-type strains N2 and CB4856. Gene expression was analyzed by microarray assays, and proteins were quantified using stable isotope labeling by amino acids in cell culture. Among all transcribed genes, we found 1,532 genes to be differentially transcribed between the two wild types. Of the total 3,238 quantified proteins, 129 proteins were significantly differentially expressed between N2 and CB4856. The differentially expressed proteins were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlates more strongly than protein abundance versus transcript abundance within each wild type. Our findings indicate that in C. elegans only a fraction of the changes in protein abundance can be explained by the changes in mRNA abundance. These findings corroborate with the observations made across species. Natural genetic variation in gene expression shapes the diversity in phenotypic traits and is the raw material for evolutionary processes (1Oleksiak M.F. Churchill G.A. Crawford D.L. 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We found 129 proteins to be significantly differentially expressed between N2 and CB4856 with at least a 1.3-fold change in abundance and a p value < 0.000457. They were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlated more strongly than the protein abundance versus transcript abundance within each wild type. The two C. elegans wild-type strains N2 (Bristol) and CB4856 (Hawaii) were grown at 20 °C on 9-cm NGM agar plates without peptone (3 g/liter NaCl, 20 g/liter bacto-agar, 5 mg/liter cholesterol, 25 mm K2PO4, 1 mm MgSO4, 1 mm CaCl2) and with a lawn of Escherichia coli (OP50 strain) bacteria. For RNA isolation we used a Maxwell® 16 AS2000 instrument with a Maxwell® 16 LEV simplyRNA tissue kit (both from Promega Corp., Madison, WI). The mRNA isolation was preceded by a modified lysis step. In short, 200 μl of homogenization buffer, 200 μl of lysis buffer, and 10 μl of a 20 mg/ml stock solution of proteinase K were added to each sample. The samples were then incubated for 10 min at 65 °C, 1000 rpm in a Thermomixer (Eppendorf, Hamburg, Germany). After cooling on ice for 1 min, the samples were pipetted into the cartridges, and the protocol as recommended by Promega was continued. After mRNA isolation, the "Two-color Microarray-based Gene Expression Analysis, Low Input Quick Amp Labeling" protocol, version 6.0, was followed, starting from step 5 (4Volkers R.J. Snoek L.B. Hubar C.J. Coopman R. Chen W. Yang W. Sterken M.G. Schulenburg H. Braeckman B.P. Kammenga J.E. Gene-environment and protein-degradation signatures characterize genomic and phenotypic diversity in wild Caenorhabditis elegans populations.BMC Biol. 2013; 11: 93Crossref PubMed Scopus (38) Google Scholar, 30Snoek L.B. Sterken M.G. Volkers R.J. Klatter M. Bosman K.J. Bevers R.P. Riksen J.A. Smant G. Cossins A.R. 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For processing the data of the RNA microarrays, the "Limma" package for the "R" environment was used. No background correction of the RNA-array data was performed as recommended by Ref. 51Zahurak M. Parmigiani G. Yu W. Scharpf R.B. Berman D. Schaeffer E. Shabbeer S. Cope L. Pre-processing agilent microarray data.BMC Bioinformatics. 2007; 8: 142Crossref PubMed Scopus (150) Google Scholar. For the "within-array normalization of the RNA-array data" the Loess method was used, and for the "between-array normalization" the Quantile method was used. The obtained normalized intensities were used for further analysis. E. coli AT713 strain (lysine/arginine auxotrophic; E. coli Genetic Stock Center, CGSC number 4529, Yale) was grown in M9 Minimal Salts Medium (30.0 g of Na2HPO4, 15.0 g of KH2PO4, 2.5 g of NaCl, 5.0 g of NH4Cl, H2O to 1 liter) supplemented with 150 mg/liter of either light (Arg-0, Lys-0) or heavy amino acids (Arg-10, Lys-8; Cambridge Isotope Laboratories). The cultures were kept for 2 days at 37 °C on a Lab-Therm Kühner orbital shaker at 230 rpm with a shaking diameter of 5 cm and harvested at an A600 between 1.5 and 3.0. Bacteria were pelleted at 1,800 × g for 15 min at 4 °C; the supernatant was aspirated, and aliquots of 50 ml were frozen at −20 °C. Adult N2 and CB4856 worms were bleached, and ∼20,000 larval stage 1 animals were transferred to NGM plates freshly seeded with light or heavy labeled bacteria at 20 °C. For the first biological replicate, CB4856 animals were fed with heavy labeled AT713. For the second and third replicates, N2 animals were fed with heavy labeled AT713. Nematode populations were grown for two generations, and proteins were isolated from animals at L4. To determine the labeling efficiency, N2 heavy protein extracts were analyzed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). The mgf files were searched against the C. elegans 6,239 database (07/03/2010, database (07/03/2010, Functional Genomics Center Zurich), using the Mascot software. The library was downloaded from the UniProt database, contains 24,362 entries, and was supplemented in-house with 259 common MS contaminants. In total, 3,908 assigned peptide spectrum matches were analyzed with a score higher than 30, which yielded 1,562 peptides. Of these, 1,552 (99.6%) peptides were heavy labeled. To extract proteins, worm samples were homogenized with glass beads (G1277 acid-washed beads, diameter of 212–300 μm, Sigma-Aldrich) in freshly prepared cell lysis buffer (8 m urea, 5% 1 m Tris-HCl, pH 8.3) in a ratio of 1:1:2 (worms/beads/buffer) at 4 °C for 30 s at 5 m/s four times (FastPrep®-24, MP Biomedicals). The lysates were centrifuged three times at 20,000 × g for 10 min at room temperature to remove debris. Protein concentration in the supernatant was determined using the Bradford reagent (Sigma-Aldrich), and 1 μg of each protein sample was checked for equimolarity by SDS-PAGE. The L4 animal lysates of N2 and CB4856 were combined in a 1:1 ratio. Protein disulfide bridges were reduced with 5 mm dithiothreitol (DTT) at 60 °C for 30 min and alkylated with 15 mm iodoacetamide in the dark at 37 °C for 1 h. 600 μg of proteins were digested with trypsin (modified sequencing grade porcine, Promega) in a ratio of 1:50 w/w overnight at 37 °C. Samples were kept frozen at −20 °C until further processing. Peptide samples were dried in a SpeedVac concentrator and resuspended in solvent A (5% ACN, 20 mm K2HPO4, pH 11) to a final urea concentration below 2 m. The pH was adjusted to 11 with 20% KOH, and the peptides were loaded on a 150 × 4.6-mm YMC Triart C18 column filled with 5 μm of silica beads (YMC Europe GmbH) at a flow rate of 1 ml/min using an Agilent 1100 liquid chromatography system (Agilent Technologies Inc.). Peptides were separated by a linear gradient from 2 to 50% of solvent B (50% ACN, 20 mm K2HPO4, pH 11) within 50 min. In total, 47 fractions were collected and pooled into 10 fractions based on the intensity measured by a UV-light detector at 214 nm. The reproducibility of the separation was controlled by running twice a self-mixed standard peptide mixture (adrenocorticotropin(1–16), angiotensinogen(1–14), bradykinin(2–9), leu-enkephalin, substance P, and vasopressin). Pooled fractions were vacuum-centrifuged until complete dryness and resuspended in 50 μl of 5% ACN, 0.1% TFA. Salts were removed using ZipTip C18 pipette tips (Millipore Corp.), and the peptides were eluted with 15 μl of 60% ACN, 0.1% TFA. Samples were dried and re-dissolved in 12 μl of 3% ACN, 0.1% formic acid to a final peptide concentration of 0.4 μg/μl. LC-MS/MS was performed on a reversed-phase nano-LC system (Eksigent) at pH 3. Peptides were separated on a self-packed reverse-phase column (75 μm × 10 cm) packed with C18 beads (Magic C18, AQ, 3 μm, 200 Å, Bischoff GmbH, Leonberg, Germany) at a flow rate of 200 nl/min. The column was equilibrated with 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1% formic acid in ACN). Peptides were eluted using the following gradient: 0–1 min; 5–9% B, 1–56 min; 9–40% B, 56–60 min; 40–50% B and 60–64 min; 50–9
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