Methods
Materials
The polyclonal chicken antibody directed against the 1- and 1-subunit of the rat lung soluble guanylyl cyclase (sGC) was from Alexis GmbH (Grünstadt, Germany); the rabbit-anti-chicken antibody was from Biogenes (Berlin, Germany). The monoclonal HuR (3A2) antibody was from SANTA CRUZ Biotechnology (Santa Cruz, 69115 Heidelberg, Germany). The oligonucleotides for RT-PCR, RNA degradation assays, and RNA-electrophoretic mobility shift analysis (RNA-EMSA) were synthesized by BioSpring GmbH (65760 Eschborn, Germany) and MWG-Biotech (85560 Ebersberg, Germany), respectively. The glutathione-agarose was from Sigma (82024 Taufkirchen, Germany). The Coomassie®Protein Assay Reagent (Prod. 23200) was purchased from Pierce Perbio (53113 Bonn, Germany), Trizol®Reagent from InvitrogenTM life technologies (76131 Karlsruhe, Germany), agarose from Roth (76185 Karlsruhe, Germany), Tween 20 from Serva (Heidelberg, Germany), and sodium chloride from Applichem (Darmstadt, Germany). All other chemicals were bought from Roth (Karlsruhe, Germany). The plasmid construct pGEX-HuR was kindly provided by Dr. Hartmut Kleinert, Mainz, Germany 1.
Animals
Investigations were performed with isolated aortic rings from 2 month old prehypertensive and aged (15-18 month) hypertensive (SHR) male rats and normotensive age-matched Wistar Kyoto rats (WKY) (n = 4 - 6 in each group). The SHR and WKY rats were kindly provided by Dr. W. Linz (Aventis Pharma Deutschland GmbH). Animal care and treatment was conducted according to institutional guidelines, in compliance with German laws and policies. The rats were held on a 12 h dark/light cycle and obtained standard chow (Altromin) and water ad libitum. Systolic blood pressure was measured in conscious rats by tail plethysmography under light anesthesia.
Preparation of the total protein from nuclear and cytosolic extracts
The thoracic aortae from young and old SHR and WKY rats were isolated from anaesthetised rats (200 mg/kg ketamin (Exalgon), 100 mg/kg xylazin (Rompun), and cleaned from fat and connective tissue. The endothelium was removed by gentle forcing and rolling a glass rod through the lumen. Thereafter the aortic tissue was snap-frozen in liquid nitrogen and stored at –70°C. To prepare nuclearand cytosolic extract a modified method described by Schreiber 2 was used. After the extraction both protein fractions were mixed to generate total native protein extracts.
Construction and purification of the GST-HuR fusion protein
The plasmid construct pGEX-HuR (generated from pGEX2T) was prepared as described 1. For the amplification and purification of the GST-HuR fusion protein an overnight culture of E. coli K12, transformed with pGEX-HuR, was diluted in 1:50 LB medium containing ampicillin (100 µg/ml). At an A600 of 0.4 the culture was induced with IPTG (0.1 mmol/L). After 5 to 6 hours of further growth, the culture was centrifuged (1 min, 13.000 rpm) and mixed in 2ml of buffer A (50 mmol/L Tris pH 8, 200 mmol/L NaCl, 1 mmol/L EDTA). Afterwards the bacterial cells were lysed by adding lysozyme (1 mmol/L) and 1% Triton for 30 min at 4°C. The lysate was spun down (13,000 rpm, 30 min) and the resultant supernatant was loaded onto a glutathione-agarose affinity column (binding capacity: 5 - 10 mg of GST/ml of resin) over the night. On the other day, columns were washed three times with PBS-T (10 mmol/L phosphate buffer pH 7.4, 1% Triton X-100) and the GST-HuR fusion protein was eluted with 7.5 mmol/L reduced glutathione and 50 mmol/L Tris-HCl pH 8.0. The protein content was determined by the Coomassie®Protein Assay Reagent.
Synthesis of sGC 1 and 1 3’-UTR mRNA by in-vitro transcription
Total RNA of rat aortic tissue was used as a template for RT-PCR amplification of the 3’untranslated region (3’-UTR) of sGC 1/1 cDNA regions, as described 3. The 3UTRSK1[a] (1.1 kb) template of the 3’-UTR sGC1 mRNA was prepared as described 3. For generating of the 1-UTR[b] (1.15 kb) template, sense primer (5’- T7AATGGCAGC CTTTGTGGGG-3’) and antisense primer (5’-GGTAAAAATAATTTATTGTAGATTC-3’), corresponding to positions 1901 to 1919 and 3023 to 3047 of the sGC1 cDNA were used (GenBank accession number M22562). Subsequently PCR fragments encompassing the 3’-UTR of sGC1 mRNA were synthesized bearing a T7 sequence at the 5’-end. Biotinylation of transcripts was performed with the North2SouthTM Biotin in-vitro transcription kit from Pierce (Rockford, USA).
Electrophoretic mobility shift assay (EMSA) with native extracts and purified GST-HuR fusion protein
EMSA’s and supershift assays with total native protein extracts from rat aortic tissue were essentially performed as described 3. The biotin-labeled oligo-ribo-nucleotides DR1GC3UTR2 comprising bases 3256 to 3295 to the 3’-UTR from the sGC1 mRNA and 1GC3UTR1 comprising bases 2929 to 2968 to the 3’-UTR from the sGC1 mRNA were obtained from MWG-Biotech AG (Martinsried, Germany). EMSAs were performed with 10 - 400 ng purified GST-HuR, and either 10 - 15 ng of biotin-labeled DR1GC3UTR2/ 1GC3UTR1 or 30 ng of biotin-labeled sGC1/13’-UTR mRNAs (see in vitro transcription) as described 3. Complexes were resolved by native 8% PAGE for 45h and electro-blotted onto nylon membranes (porablot NY amp, Machery-Nagel, Düren). Blocking and detection of biotin-labeled bands was performed as described 3.
In vitro RNA degradation assay
Biotin-labeled riboprobes (150ng; DR1GC3UTR2/1GC3UTR1) were incubated with 500 – 600 ng protein extract for 15, 30 and 45 min at 30°C. RNA degradation was stopped by RNA sample buffer (80% deionized formamide, 8.5% formaldehyde, 1X TBE). The degradation products were separated on a denaturing 20% polyacrylamide gel containing 7 mol/L purified urea and electro-blotted onto nylon membranes (porablot NY amp). Blocking and detection of biotin-labeled bands was performed as described 3.
Northern blots
Total RNA samples were fractionated and electro-blotted as described previously 3,4. Hybridization occurred at 42°C overnight with 5’- and 3’-end biotinylated-DNA probes specific for HuR and elongation factor II mRNA. These biotin-labeled oligonucleotides SKNORTH1HUR (5’GCG GGC TCG AGT CTC CAC TAG TTT CTG CGG3’) comprising bases 407 to 436 of the translated region from the rat HuR mRNA and SKNORTH5EFII (5’CCC GGG TGG ACT ACT GCT CCG GCG GTA CCC-3’) comprising bases 1171 to 1200of the translated region from the rat ef II mRNA were obtained from MWG-Biotech AG. Washing, blocking and detection of biotin-labeled bands was performed as described 3.
Reference List
1.Rodriguez-Pascual F, Hausding M, Ihrig-Biedert I, Furneaux H, Levy AP, Forstermann U, Kleinert H. Complex contribution of the 3'-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR. J Biol Chem. 2000;275:26040-26049.
2.Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer
binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic
Acids Res. 1989;17:6419.
3.Kloss SW, Furneaux H, Mulsch A. Posttranscriptional regulation of soluble
guanylylcyclase expression in rat aorta. J Biol Chem. 2003;278:2377-2383.
4.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.
[a] 3UTRSK1: truncated 3'UTR of sGC 1 mRNA
[b]1-UTR: truncated 3'UTR of sGC 1 mRNA