SuppLEMENTAL INFORMATION
Translational regulation of Glutathione peroxidase 4 Expression through Guanine-rich sequence binding factor 1 is essential for embryonic brain development.
Christoph Ufer1,2), Chi Chiu Wang3,4), Michael Fähling5), Heike Schiebel1), Bernd J. Thiele5), E. Ellen Billett2), Hartmut Kuhn1) and Astrid Borchert1)
1)Institute of Biochemistry, University Medicine Berlin - Charité, Monbijoustr. 2, D-10117 Berlin, F.R. Germany, 2)School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, United Kingdom, 3)Li Ka Shing Institute of Health Sciences and 4)Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong, 5)Institute of Physiology, University Medicine Berlin - Charité, Tucholskystr. 2, D-10117 Berlin, F.R. Germany
1. Methodological details
1.1. PCR primers
The sequence of the oligonucleoitides used for qRT-PCR (different purposes) are given below (Table S1).
Gene product
/ Direction / SequenceGPx4 5’UTR probe used for the yeast three hybrid screen
m-GPx4 / Forward / 5’- CAG GGG CCT CGC GTC TTA GCG-3’
Reverse / 5’- CAT CTC GGC GGC CGG AGC CA-3’
GPx4 5’UTR probe used for the RNA mobility shift assay
m-GPx4 / Forward / 5’- TAA TAC GAC TCA CTA TAG GGT ACT CAG GGG CCT CGC GTC TTA GCG-3’
Reverse / 5’- GGG GGC TAG CTC TCG GCG GCC GGA GCC AGC G-3’
Grsf1 coding sequence for mammallian expression
Grsf1 / Forward / 5’ GAA TTC GCC ATG GCC GGG ACG CGC TGG GTG CTA G-3’
Reverse / 5’ AAG CTT ATT TTC CTT TAG GAC ATG AAT TTA GGA-3’
Quantitative RT-PCR
Grsf1 / Forward / 5’- GAA TCC AAA ACT ACC TAC CTG GAA G-3’
Reverse / 5’- CAG CTG TAA GGA AGT CCT CTC AG-3’
m-GPx4 / Forward / 5’- GAG ATG AGC TGG GGC CGT CTG A-3’
Reverse / 5’- ACG CAG CCG TTC TTA TCA ATG AGA A-3’
GAPDH / Forward / 5’- CCA TCA CCA TCT TCC AGG AGC GA-3’
Reverse / 5’- GGA TGA CCT TGC CCA CAG CCT TG-3’
m-GPx4 (RNA-IP) / Forward / 5’- CGT CCA TTG GTC GGC TGC GTG
Reverse / 5’- TCC TGG CTC CTG CCT CCC AAA C
Grsf1 coding sequence for prokaryotic expression
Grsf1 / Forward / 5’- GGA TCC GGG ACG CGC TGG GTG CTA G-3’
Reverse / 5’- CCC CGC GGC CGC TTA TTT TCC TTT AGG ACA TGA ATT TAG G-3’
K6-Grsf1 / Forward / 5’- CCC CGG ATC CAT TGG GCA CGG GAA CAA GGG AC-3’
Reverse / 5’- CCC CGC GGC CGC TTA TTT TCC TTT AGG ACA TGA ATT TAG G-3’
In situ hybridization probes
Krox20 / Forward / 5’- TCA TAG GAA TGA GAC CTG GGT CCA T-3’
Reverse / 5’- AGA TGG CAT GAT CAA CAT TGA CAT GA-3’
Gbx2 / Forward / 5’- TCG GGG CTG TCC GAG GGC AAG-3’
Reverse / 5’- GCT GCT GGT GTT GAC TTC GAA TAG-3’
Grsf1 / Forward / 5’- GAA TCC AAA ACT ACC TAC CTG GAA G-3’
Reverse / 5’- CTT CTC CCT CTA TAG TCC ATC ACA A-3’
m-GPx4 (anti-sense) / Forward / 5’- AAGGCTTCGGCCTCGCGCGTCCATTGGT-3’
Reverse / 5’- TAATACGACTCACTATAGGGCACAGCAGTGCTGGCTTAAGTAAG-3’
m-GPx4 (sense) / Forward / 5’- TAATACGACTCACTATAGGGCGGCCTCGCGCGTCCATTGGT
Reverse / 5’-AAGCTTCACAGCAGTGCTGGCTTAAGTAAG
1.2. Yeast three-hybrid system
The yeast three-hybrid system is a screening method for detection of RNA binding proteins. We used this method to screen a murine testis expression library for proteins capable of binding to the 5’-UTR of the m-GPx4 mRNA. For this purpose we amplified the 5’-UTR by PCR using the primer combination indicated in Table S1 (supplemental data). PCR resulted in a 146 bp product representing the 5’-UTR. This fragment was cloned into the vector pIIIA/MS2-1 using the SmaI restriction site. The recombinant plasmid coding for the MS2 hybrid RNA and the selection markers ADE2 and URA3 was introduced into the yeast strain YBZ-1. The yeast strain YBZ-1 bears the reporter genes HIS3 and LacZ in its genome and is auxotroph for histidine, leucine, uracil and adenine. The initial transformation was followed by large-scale transformation (200 µg plasmid DNA) of the cells with a commercial mouse testis cDNA library (Clontech, Palo Alto, USA) that encodes for testicular proteins fused to an N-terminal GAL4 transcriptional activation domain and the selection marker LEU2. Double transformants were grown in a medium (deficient in essential amino acids Leu and His) selecting for activation of the reporter gene HIS3 and the presence of the of the library plasmid pACT2 as well as containing 2,5 mM 3-aminotriazol (3-AT) to suppress background growth. 3-AT is a competitive inhibitor of the HIS3 gene product. Initial selection was not for the maintenance of the RNA plasmid. Clones with RNA-independent reporter gene activation will grow independent of the plasmid pIIIA/MS2-1 and eventually lose it. Since this plasmid confers ADE2 prototrophy, RNA-independent clones that lost the plasmid will start de novo synthesis of adenine once the adenine in the medium becomes low. This leads to accumulation of a red purine metabolite due to the lack of the ADE2 gene product and thus renders yeast colonies pink. From 107 transformants about 1300 white clones (His+ and Leu+ prototrophes) were selected for further analysis. First, activation of the second reporter gene ß-galactosidase was assayed by direct measurement of enzyme activity using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). About 95% of the initial clones also activated the second reporter gene. To see whether the reporter gene activation is dependent on the hybrid RNA these yeast clones were subjected to two rounds of URA3 counter selection. Clones were cured of the pIIIA/MS2-1 plasmid in the presence of Uracil and 0.1% 5-fluoroorotic acid (5-FOA) and activity of the reporter gene ß-galactosidase was assayed for. The URA3 gene product converts 5-FOA to toxic 5-fluorouracil. Thus only clones lacking the plasmid pIIIA/MS2-1 will grow in the presence of 5-FOA. Only 2% of these clones were truly RNA-dependent. Into the remaining clones control plasmids (pIIIA/MS2-1 lacking the m-GPx4-5’UTR or expressing the GPx4-5’UTR in the reverse-complementary orientation) were introduced for determining sequence specificity by using mating assay. For this purpose the control plasmids were introduced into the yeast strain R40-coat (MATa). These clones were mated to the YBZ-1 (MATa) clones resulting from the previous URA3 counter selection being devoid of the plasmid pIIIA/MS2-1. Diploid cells were selected on selection medium (-Leu, -His, -Ura) for the presence of both plasmids (pACT2 and pIIIA/MS2-1) and activation of the reporter gene HIS3 and activity of ß-galactosidase was tested. Eventually, specificity of reporter gene activation was confirmed by co-transformation experiments and selection on selection medium (-Leu, -His, -Ura) in the presence of 80 µg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside by colony growth and color.
1.3. 5’-UTR-driven reporter gene assays
To explore the impact of Grsf1 on translation of the m-GPx4 mRNA, UTR-dependent reporter gene assays were carried out. For this purpose a modified pGL3-promoter vector (Promega, Mannheim, Germany) was used. The vector specific 5’-UTR of luciferase mRNA was replaced by the GPx-4 5’-UTR using HindIII (5’-end) and NcoI (3’-end) restriction sites. For the deletion experiments the AGGGGA motif was removed by PCR. For Grsf1 expression in mammalian cells the coding region of Grsf1 was cloned into pcDNA3.1(-) (Invitrogen, Karlsruhe, Germany) using primers as specified in table 1 (supplemental data) containing restriction sites for EcoRI and HindIII respectively. The structures of all modified vectors were confirmed by DNA sequencing. Mouse embryonic fibroblast (mEF ras/TAg) cells (Ryan et al. 2000) were maintained in Dulbecco’s modified Eagle’s medium (high glucose; PAA Laboratories GmbH) supplemented with 10% heat-inactivated fetal calf serum, 50U/ml penicillin, 50 µg/ml streptomycin, 15 mM Hepes and 2 mmol/l glutamine at 37°C, 5% CO2 in 96-well plates (µClear Platte 96K, Greiner BIO-ONE GmbH). Before use cells were maintained in a medium containing 0.4% fetal calf serum for at least 24 h. Cells were co-transfected with the modified Firefly-luciferase pGL3-promoter vector (Promega, Mannheim, Germany) and the Renilla-luciferase phRL-TK vector (transfection control) using the MagnetofectionTM PolyMag transfection system (OZ Biosciences, Marseille, France) according to the manufacture’s instruction. Following transfection (20 min on a magnetic plate) the transfection medium was replaced with fresh medium containing 10% heat-inactivated fetal calf serum. Co-transfection with an expression vector encoding the Grsf1 trans-factor was used in a ratio 1:2.5 (Firefly-luciferase vector : Grsf1-expression vector). In order to avoid differences in the amount or ratio of the constitutive CMV-promoter in the kinetic study we successively replaced the empty expression vector (backbone vector) with the expression vector encoding the Grsf1 protein. Luciferase activity was detected after 6 h using the Dual-GloTMLuciferase Assay System (Promega, Mannheim, Germany) and a luminometer (Labsystems Luminoscan RS) programmed with individual software (Luminoscan RII, Ralf Mrowka). Results are given as means ± SD. Data were analyzed using the Student’s t-test, and null hypothesis was rejected at the 0.05 level.
1.4. Quantitative RT-PCR (qRT-PCR)
qRT-PCR was carried out with a Rotor Gene 3000 system (Corbett Research, Mortlake, Australia) using the QuantiTect SYBR Green PCR Kit from Qiagen (Hilden, Germany). The following PCR protocol was applied (Borchert et al. 2006): 15 min hot start at 95 °C, followed by 40 cycles of denaturation (30 sec at 94°C), annealing (30 sec at 65°C) and synthesis (30 sec 72°C) in a total volume of 10 µl. Homogeneity of the amplified PCR products was tested recording the melting curves. For this purpose the temperature was elevated slowly from 60°C to 99°C. Amplification kinetics were recorded in the real-time mode as sigmoid process curves, for which the fluorescence was plotted against the number of amplification cycles. To generate standard curves for exact quantification of the expression levels, specific amplicons were used as external standards for each target gene. The initial amplicon concentrations varied between 5 x 103 and 3 x 106 copy numbers. GAPDH mRNA was used as internal standard to normalize expression of the target transcripts (Grsf1 and m-GPx4). Absolute ratios of the target mRNA species and the GAPDH mRNA were calculated using these standard curves. All RNA preparations were analyzed at least in triplicates and means ± SD are given. The experimental raw data obtained during amplification were evaluated with the Rotor-Gene Monitor software (version 4.6).
1.5. Recombinant expression of Grsf1
Grsf1 was expressed in E. coli as a glutathione transferase (GST) fusion protein. It was purified from the bacterial lysate supernatant by affinity chromatography on a glutathione agarose column. For this purpose the coding regions of murine Grsf1 (Nm_178700) and K6-Grsf1 (splicing variant) were amplified using the primers specified in table 1 (supplemental data) containing the recognition sequences for BamHI (5’-primer) and NotI (3’-primer). The PCR products were cloned into the plasmid pGEX-4T-3 (Amersham, Freiburg, Germany) using the Bam HI and Not I restriction sites present in the vector. The resulting plasmids pGEX-4T-3/Grsf1 and pGEX-4T-3/K6-Grsf1 were then transformed into E. coli (strain BL21, Amersham, Freiburg, Germany). Expression was induced with 0.5 mM isopropyl a-D-thiogalactoside and bacteria were incubated at 30° C for 1 h. Cells were lysed by sonication and the 10,000 g lysis supernatant was used as starting material for affinity chromatography on a glutathione-agarose column (Sigma, Deisenhofen, Germany). GST-tagged recombinant proteins were eluted from the column using a step gradient of reduced glutathione reaching a final concentration of 10 mM in 50 mM Tris-HCl, pH 9,5.
1.6. RNA immunoprecipitation
In order to detect in vivo RNA/protein interactions RNA immunoprecipitation was performed. In brief, 2x106 murine neuroblastoma N2a cells (maintained in 90% DMEM, 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 10 µg/ml streptomycin (Sigma, UK) at 37°C in a humidified atmosphere of 95% air/5% CO2) were washed twice with phosphate buffered saline (PBS) and fixed in 1% formaldehyde for 20 min at room temperature. The reaction was stopped with 150 mM glycine, cells were washed twice with PBS, resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 80 u/ml RNasin (Promega, Southampton, UK) and EDTA-free proteinase inhibitor mini cocktail (Roche Diagnostics Ltd., UK) was added. After sonication lysates were cleared by centrifugation at 16,000g for 15 min at 4º C followed by the addition of 60 ml protein A or G agarose (Upstate, CA, USA) to the supernatant and subsequent agitation at 4oC for 3 hours. Agarose beads were then pelleted by low speed centrifugation and the pre-cleared supernatants were incubated over night at 4oC with 10 mg of a polyclonal anti-Grsf1 antibody (Abcam, Cambridge, UK), monoclonal anti-FLAG M2 antibody (Sigma, UK), polyclonal anti-mouse immunoglobuline (Dako, Denmark) or without antibody in the presence of 50 u/ml SuperRNAseIN (Ambion, Huntingdon, UK). Immune complexes were precipitated by addition of 60 ml protein A or G agarose and low speed centrifugation. Precipitates were washed once with 1 ml of each of the following buffers in the presence of 50 u/ml SuperRNAsIN (Ambion, Huntingdon, UK): RIPA buffer; low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH8.0]); high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH8.0]); LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris [pH8.0]) and twice with TE buffer (10 mM Tris-HCl [pH8.0], 1 mM EDTA). Finally protein/RNA complexes were eluted with 250 ml elution buffer (100 mM Tris-HCl [pH 7.8], 10 mM EDTA, 1% SDS). Eluates and an aliquot of cleared lysate (“Input”) were adjusted to 100 mM NaCl and incubated with 20 mg proteinase K (Ambion, Huntingdon, UK) at 42º C for 1 hour and at 65ºC for 1 hour followed by RNA extraction using TRI Reagent (Ambion, Huntingdon, UK) according to the vendor’s instructions. After DNA digestion (TURBO DNA-free kit from Ambion) RNA was reversely transcribed with SuperScript II (Invitrogen) following standard protocols. The resulting cDNAs were amplified and quantified using a quantitative PCR approach as described above. The primer sequences are given in the Supplemental Information, Table S1.
2. Supplemental experimental data
2.1. Co-transformation experiments confirm specific binding of Grsf1 to the m-GPx4 5’UTR in yeast
High binding specificity as revealed by the yeast three hybrid screen was confirmed by co-transformation experiments (Fig. S1 A, B), which indicated that activation of the lacZ reporter gene (blue staining) was only observed when all hybrids were correctly expressed.