Cell culture
Human embryonic kidney (HEK) 293T cells were cultured in DMEM containing 3.7 g/l sodium bicarbonate and supplemented with 10% FBS (Gibco-BRL), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-BRL) in a 5% CO2 humidified atmosphere (37°C). Mouse RAW264.7 macrophages were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS (Gibco-BRL), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-BRL) in a 5% CO2 humidified atmosphere (37°C).
Plasmid constructs
The plasmids for TTP, Zfp36l1, Zfp36l2 and 14-3-3ζ expression were constructed as described [15, 16, 21, 29]. The TTP mutants S316 and S318, S52 and S178 were created by PCR (for S316 and S318) or Q5 site-directed mutagenesis kit (New England Biolabs) (for S52 and S178) using the primers indicated in Table S1. The PCR products were ligated to pCMV-Tag2 (Stratagene) and pEGFP-C2 (Clontech) for mammalian cell expression. Mouse Cnot1@800-1310 were PCR amplified from a cDNA template (OriGene) with the primers: 5’-CAGGCTCAGGCCCAGGTT-3’ and 5’-TTATTAGGCCTGAGCCAGTGCAATAC-3’. The PCR products were cloned into pGEX4T-1 for expression of glutathione S-transferase (GST)-fused proteins in bacteria.
RNA isolation, reverse transcription, and quantitative PCR
Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen). For mRNA stability analysis, the cells were treated with 10 µg/ml actinomycin D (transcription inhibitor) for various times to inhibit new transcription. After DNase I digestion, 2 µg total RNA was reverse-transcribed to produce cDNA using M-MLV reverse transcriptase and oligo dT primer (Promega). Real-time PCR was performed with the 7300 Real-Time PCR System (Applied Biosystems) in a total volume of 20 μl. Expression of genes encoding TTP, TNFα and actin was assessed using SYBR Green PCR Master Mix (Applied Biosystems) with 50 ng of cDNA and 160 nM of each primer: 5-GGATCTCTCTGCCATCTACGA-3 and 5-CAGTCAGGCGAGAGGTGAC-3 for TTP; 5’-GACCCTCACACTCAGATCATCTTCT-3’ and 5’-CCTCCACTTGGTGGTTTGCT-3’ for TNFα; 5-TCCTTCCTGGGCATGGAGTC-3 and 5-ACTCATCATACTCCTGCTTG-3 for β-actin. The PCR amplification conditions were 40 cycles of 95°C for 15 s and 60°C for 1 min. Real-time PCR data were analyzed using the 2–CT relative quantitation method.
Cell extracts preparation, co-immunoprecipitation (IP), and western blotting
293T cells were cotransfected with the plasmids using Turbofect reagent (Thermo). Cells harvested 24 h after transfection were lysed with NET buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.1% (v/v) Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (10 mM β-glycerol phosphate; 0.1 mM Na2MoO4; 0.1 mM Na3VO4, pH 10.0; 10 mM NaF) and centrifuged at 15,000 × g for 10 min. The supernatants were immunoprecipitated using anti-Flag M2 agarose (Sigma-Aldrich) at 4°C for 2 h. After the IP mixture was washed three times with NET buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Millipore), and western blotting was performed using anti-Flag (Sigma-Aldrich), anti-HA (Bethyl), anti-TTP (produced in our lab), anti-α-tubulin, anti-β-actin, anti-phospho-ERK1/2, anti-p38, anti-phosph-p38, anti-MK2, anti-phospho-MK2, anti-phospho-RSK1, anti-CNOT3 and anti-CNOT6 (all from Cell signaling), anti-GAPDH, anri-CNOT1, and anti-CNOT7 (all from Proteintech Group), anti-ERK1/2 and anti-RSK1 (both from Santa Cruz Biotechnology), and anti-DDX6 (abcam). All experiments were carried out at least three times, and represented results were displayed.
Generation of rabbit anti-phospho-S316 of TTP
A peptide containing the sequence surrounding phospho-S316 of TTP (RLPIFNRIpSVSE) was synthesized and purified. A specific rabbit antiserum was produced by LTK BioLaboratories (Taiwan). The antiserum was affinity purified using the immunizing peptide (LTK BioLaboratories).
In vitro kinase assay
Each reaction mixture contained 2 µg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 µl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha (SignalChem), RSK1 (SignalChem), and MK2 (SignalChem) in a final volume of 30 µl. The kinases can be from cell extracts. 300 g of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 g of GST-TTP in the buffer containing 20 mM HEPES, pH7.7, 75 mM NaCl, 0.5 mM MgCl2, 0.1 mM EDTA, 0.05 % Triton X-100, 0.5 mM DTT, 20 mM -glycerolphosphate, 0.1 mM Na3VO4, 1g/ml leupeptin, 1g/ml papstatin A, 100 g/ml PMSF. The mixture was rotated at 4oC for 3 hr and pelleted by centrifigation at 10,000xg for 20 sec. After 4x1-ml washes in HEPES binding buffer (20 mM HEPES, pH7.7, 50mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05 % Triton X-100), the beads were resuspended in 30 l for kinase assay. The reaction mixtures were incubated at 30°C for 30 minutes and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.
GST pull-down assays
Glutathione-Sepharose 4B (~8 µl, GE Healthcare Life Sciences) were incubated with 2 μg of bacterially expressed GST, or GST-Cnot1@800-1015 or GST-14-3-3 in phosphate-buffered saline containing 1% (v/v) Triton X-100 on a rotary shaker for 20 min at room temperature. After washing three times with the same buffer, the Sepharose was combined with lysates (300 µg protein) of RAW264.7 cells that had undergone various treatments in a final volume of 200 µl of buffer containing 20 mM HEPES, pH 7.9, 100 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% (v/v) NP-40, 1% (v/v) Triton X-100, 1 mM DTT, and 1 mM PMSF. The mixtures were incubated at 4°C for 2 h on a rotary shaker, and then the Sepharose was washed four times with the same buffer lacking DTT and PMSF but containing 0.2 M NaCl and once with 50 mM Tris, pH 6.8. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by western blotting.
RNA pull-down assays
Cytoplasmic extracts from LPS-stimulated RAW264.7 cells were prepared by hypotonic buffer (10 mM HEPES, pH 7.5, 10 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 mM DTT, 0.05% NP-40, and protease inhibitor cocktails). Potassium acetate was adjusted to 90 mM, and 0.1 U·μL-1 RNasin (Promega, Madison, WI) and 20 μg·μL-1 yeast tRNA were added to each lysate. To prevent non-specific binding, heparin-agarose (Sigma-Aldrich) was incubated with each lysate for 15 min at 4°C and then centrifuged for 1 min at 8,000 rpm, 4°C. Each supernatant was further cleaned with streptavidin-Sepharose (8 µL; Invitrogen) for 1 h at 4°C and then centrifuged for 1 min at 8,000 rpm, 4°C. The biotin-labeled TNFα ARE was added as described [21]. The pulled-down RNA-protein complexes were washed four times with binding buffer (hypotonic buffer containing 90 mM potassium acetate) and separated by SDS-PAGE (10% acrylamide) for western blotting analysis.
siRNA-mediated knockdown, transfection and immunoprecipitation assay
HEK293T cells (1 105) were seeded in each well of a 12-well plastic culture plate. For gene knockdown, the cells were transfected with 5 nM of a small interfering RNA (siRNA) for CNOT1, CNOT6, CNOT7, RSK1 or MK2 (Invitrogen) using Lipofectamine 3000 (Invitrogen). After 24 h, the cells were transferred into fresh medium and transfected with 1 µg of Flag-TTP wild-type, -TTP S52,178A or –TTP S316A expression plasmids using Turbofect reagent (Thermo). After another 24 h, cells were harvested, and whole cell extracts were isolated for western blotting or immunoprecipitation.
CRISPR/Cas9-mediated gene editing
CRISPR-based sgRNAs were designed on Benchling (https://benchling.com) and CHOPCHOP (http://chopchop.cbu.uib.no/) to search the specific target-sequences of Cas9 RNP complexes on mouse Ttp gene. Based on the description of target score [30] (http://crispr.mit.edu/about), we designed four sgRNAs, which contain a relatively higher on-target with a lower off-target score (Table 3) . Each DNA template of Ttp sgRNA encoding for a T7 promoter, a 20 nt target sequence and a published sgRNA scaffold [31] were assembled by overlapping PCR. Each PCR reactions contain 20 nM premix of TTP sgRNAs and bottom scaffold (Table S2), 1 μM premix of T7 oligo primer and sgRNA-reverse, 200 μM dNTP and Q5 polymerase (NEB) according to manufacturer's protocol. The thermocycler setting consisted of 30 cycles of 95°C for 10 seconds, 59°C for 10 seconds and 72°C for 10 seconds. [32].
The assembled PCR products were extracted once with phenol:chloroform:isoamylalcohol and then once with chloroform, before isopropanol precipitation overnight at −20°C. The DNA pellet was washed three times with 70% ethanol and dissolved in DEPC-treated water. The T7 in vitro transcription reaction consisted of 30 mM Tris–HCl (pH 7.9), 20 mM MgCl2, 0.01% Triton X-100, 2 mM spermidine, 10 mM DTT, 5 mM of ribonucleotide triphosphate, 100 μg/ml T7 polymerase and 1 μM DNA templates. The reaction was incubated at 37°C for 4 hr, and RNase-free DNase was added to digest the DNA template 37°C for 1 hr. The reaction was stopped by adding 2xSTOP solution (95% deionized formamide, 0.05% bromophenol blue and 20 mM EDTA) at 60°C for 5 min. The RNA was purified by electrophoresis in 10% polyacrylamide gel containing 6 M urea. The RNA band was excised from the gel, grinded up in a 15-ml tube, and eluted with 5 vol of 300 mM sodium acetate (pH 5) overnight at 4°C. One equivalent of isopropanol was added to precipitate the RNA at −20°C. The RNA pellet was centrifuged and washed three times with 70% ethanol, and dried by vacuum. To refold the sgRNA, the RNA pellet was first dissolved in 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol and 1 mM TCEP. The sgRNA was heated to 70°C for 5 min and cooled to room temperature. MgCl2 was added to a final concentration of 1 mM. The sgRNA was again heated to 50°C for 5 min, cooled to room temperature and kept on ice. The sgRNA concentration was determined by OD260nm and adjusted to 100 μM using 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, 1 mM TCEP and 1 mM MgCl2. The sgRNA was store at −80°C [32].
Transfection of RAW264.7 cells were performed according to the instructions of Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Invitrogen). The Cas9 RNP were prepared by incubating the purified Cas9 protein with sgRNA at 1:4 molar ratios. After transfection, the cells were incubated at 37°C for 48 hr, and followed by single cell sorting using BD, FACSJazz automated cell sorter to perform the single isolation. (TechComm at NTU)
Genomic DNA isolation and PCR analysis
Genomic DNA was extracted by QuickExtract™ DNA Extraction Solution (epicentre). Cells were mixed by 200 µl of QuickExtract Solution and vortex for 15 seconds. The tubes were transferred to 65°C and incubate for 6 minutes. After 15 seconds vortex, samples were incubated at 98°C for 2 minutes. Store the DNA at –20°C, or at –80°C for long term storage according to manufacturer's protocol. Genomic DNA were PCR amplified using primer-1 and primer-4 (Table S2). Theoretically, sgRNA mTTP KO-1 and sgRNA mTTP KO-4 will generate double-stand DNA break on each of the target site, and cause a 793 nt deletion between exon 1 and exon 2 on Ttp gene in RAW264.7 cells. Therefore, the size of PCR product generated by primer-1 and primer-4 will be reduced from 2001 nt to 1208 nt. Besides, primer-2 and primer-3 locate on the predicted cutting fragment. If the TTP knock-out happened, it would not have PCR products between primer-1 and primer-2 or primer-3 and primer-4 by theoretically (Table S2).
Statistical analysis
All data are presented as the mean ± SD of at least three independent experiments. Statistical significance (*P < 0.05, **P < 0.01 or ***P < 0.001) was determined by one-tailed Student's t-test.