Cell lines and cell transfection
293T and Hela cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, in a 37°C, 5% CO2 incubator. Cells were transfected using the standard calcium phosphate precipitation method.
Plasmids pTip110.His, pTip110s.His, and deletion mutants ∆NLS and ∆RRM, and pGEX-Tip110, pGEX-Tip110s, and deletion mutants ∆NLS and ∆RRM, and pAD.Tip110 were described elsewhere . The standard PCR cloning technique was used to construct pTip110∆LSM, with pTip110.His as the templates and primers 5′-GGA ATT CAC CAT GGC GAC TGC GGC CGA A-3′ and 5′-CCG CTC GAG TCA ATG ATG ATG ATG ATG ATG GGC AAC TGC AGG AGC CG-3′ (the restriction enzyme sites underlined). The Tip110 siRNA expression plasmid was constructed in the backbone of pSHAG-1 by annealing oligonucleotides 5′-CGG
GAT CCG ACT CAG CCT CGG GTT CTG AA-3′ and 5′-CGG GAT CCA AAA AAT TGG ACT CAG CCT CA-3′, and inserting the annealed DNA at the Bam HI site of pSHAG-1. All recombinant plasmids and deletion mutants were verified by sequencing. The sources of the other plasmids used in the study are: pGEX-4T-3 from Amersham (Piscataway, NJ, USA), pSHAG-1 from Dr. Gregory Hannon, pIIIA.MS2 from Dr. Marvin Wickens, pT7.U6 from Dr. Iain Mattaji, pDM138 from Dr. Thomas Hope, pcRev from Dr. Bryan Cullen, and pSP64-Hβδ6 from Dr. Adrian Krainer.
Construction of a hybrid RNA library and yeast three-hybrid screening
A hybrid RNA library and yeast three-hybrid screening were performed as previously described , with some modifications. Briefly, genomic DNA was isolated from 293T cells and digested by Mse I, Tsp509I, Alu I and Rsa I, and then by T4 DNA polymerase in the presence of 100 μM dNTP. Digested DNA was fractionated on a 2% agarose gel to obtain DNA fragments of 50–150 bp. These small DNA fragments were then ligated into SmaI-linearized and calf intestine phosphatase-treated pIIIA.MS2 plasmid. The ligation mixture was transformed into GC5 competent bacteria for propagation and isolation of the library DNA. Yeast strain L40coat stably expressing LexA-MS2 coat fusion protein was first transformed with pAD-Tip110 plasmid and then with the RNA expression library. The selection of transformed yeast was performed on synthetic complete plates containing 0.5 mM 3-aminotriazole and no tryptophan, leucine, uracil and histidine, followed by X-gal filter assay for β-galactosidase expression. β-galactosidase-positive colonies were further cultured to isolate the hybrid RNA plasmids.
Recombinant protein purification
GST-Tip110, GST-Tip110s, Tip110 deletion mutants ∆NLS and ∆RRM, and GST proteins were expressed in and purified from E. coli BL21, as previously described . Briefly, expression plasmids were transformed into BL21 and induced with 1 mM isopropyl-d-thiogalactopyranoside for 2 h at 37°C. GST fusion proteins were purified using a Pierce GST fusion protein purification kit (Rockford, IL, USA). When necessary, GST was removed by treating the eluted protein with thrombin protease (10 units) (Invitrogen, Carlsbad, CA, USA) at room temperature for 18 h. The digested protein solution was dialyzed overnight in 4 l of phosphate-buffered saline and cleared of GST protein by additional incubations with fresh glutathione beads. The purified proteins were electrophoresed on a 8% SDS-polyacrylamide gel and stained with Gold–Blue (Pierce) to ensure the complete removal of GST protein as well as undigested fusion protein and other contaminated proteins.
RNA–protein gel mobility shift assay
U6 RNA was synthesized using an Ambion in vitro transcription kit (Austin, TX, USA). Briefly, pT7-U6 plasmid was linearized with Dra I and incubated with ATP, CTP, GTP and 32P-labeled UTP in the presence of T7 RNA polymerase at 30°C for 1 h, followed by removal of free nucleotides by an CentriSpin column (Princeton Separation, Princeton, NJ, USA). 32P-labeled-U6 RNA was then mixed with purified GST-Tip110 or its mutant proteins in a total volume of 20 μl containing 1 mM HEPES, pH 7.6, 1 μM MgCl2, 16 mM KCl, 2.5% glycerol (v/v), and 10 nM DTT. The mixture was incubated on ice for 15 min and then subjected to 5% nondenaturing polyacrylamide gel electrophoresis (acrylamide-bisacrylamide, 80:1) in the 0.5× TBE buffer. The gel was dried and exposed for autoradiography.
Preparation of pre-mRNA and in vitro splicing assay
Human β-globin minigene pre-mRNA was synthesized using an Ambion in vitro capped RNA transcription kit (Ambion). Briefly, pSP64-Hßδ6 plasmid was linearized with BamH I and incubated with ATP, CTP, GTP, cap anolog and 32P-labeled UTP at 37°C for 2 h. 32P-labeled pre-mRNA was then purified on a 5% denaturing polyacrylamide gel and suspended in 10 mM Tris.HCl, pH 7.9. The 32P-labeled pre-mRNA was then aliquoted and stored at −80°C. Splicing reactions were carried out in a 25 μl volume containing 12.8 μM MgCl2, 500 μM ATP, 20 mM creatine phosphate, 2.7% (w/v) polyvinyl alcohol, 1,000 U/ml RNasin, 12.8 mM HEPES, pH 8.0, 14% (v/v) glycerol, 62 mM KCl, 0.12 mM EDTA, and 0.7 mM DTT at 30°C for 2 h. The reactions contained 6 μl Hela nuclear extract (Promega, Madison, WI, USA) and 12.5 ng 32P-labeled pre-mRNA prepared above.
Monoclonal antibody production
Anti-Tip110 monoclonal antibodies were produced by Promab Biotechnologies, Inc. (Albany, CA, USA). Briefly, purified GST-Tip110 protein was used to immunize BALB/c mice. The mice were given an additional boost immunization after 3 weeks. Three days after the boost, the spleen cells were isolated from the immunized mice and fused with SP20 cells (ATCC) using polyethylene glycol (Sigma, St. Louis, MO, USA). Resulting hybridoma were screened to be specific anti-Tip110 antibody using ELISA. To purify the monoclonal antibody, the hybridoma cells were cultured in a production module of the miniPERM device (Viva Science, Inc.), and the antibody was further purified from the culture supernatant using a Protein A column and a AKTA prime-automated liquid chromatography system (Amersham, Piscataway, NJ, USA). The antibody protein purity was determined to be IgG2 with a purity of higher than 99%.
Purified Tip110 monoclonal antibody (20 μg) was incubated with 50 μl of 50% slurry protein A-Sepharose (Amersham) in a buffer containing 20 mM HEPES, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.5 mM PMSF, 1 mM DTT at 4°C for 4 h with continuous mixing. The protein A beads were then recovered by centrifugation and repeated washes with the same incubation buffer to remove excess anti-Tip110 antibody. An aliquot of the beads were then incubated with Hela nuclear extract at 4°C for 2 h with continuous mixing, and the supernatant was used as Tip110-depleted Hela nuclear extract. Tip110 protein on the immunoabsorbed beads was eluted by adding 20 μl of 4× SDS sample buffer, followed by boiling 5 min. The eluent and aliquot of the Tip110-depleted Hela nuclear extract were subjected to 10% SDS-PAGE gel and Western blot analysis to ensure efficient Tip110 depletion.
Pre-mRNA splicing-dependent CAT reporter gene assay
293T cells were plated in a 6-well plate at a density of 0.72 × 106 cells/well and 1 day later transfected with 1 μg pDM138, 1 μg pcRev, 1–5 μg pTip110.His or pTip110.siRNA. In all transfections, a CMVβGal plasmid was included to normalize transfection variations, and pcDNA3 was used to equalize the amount of transfected DNA. Forty-eight hours after transfection, the cells were harvested and subjected to 3 rounds of freeze–thaw in 0.25 M Tris.HCl, pH 8.0, and the lysates were collected for β-galactosidase and CAT activities. β-galactosidase was determined using a colorimtric assay , while CAT was determined by one-step simple phase extraction and scintillation counting as described .
Preparation of cell lysates and Western blot analysis
Cells were washed twice with ice-cold phosphate-buffered saline (PBS), and cell pellets were suspended in two volumes of whole cell lysis buffer containing10 mM NaHPO4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium azide, 0.5% sodium deoxycholate, 0.004% sodium fluoride, and 1 mM sodium orthovanadate and incubated on ice for 10 min. Cell lysates were obtained by centrifugation, removal of the cell debris, and electrophoretically separated on 10% SDS-PAGE and analyzed by immunoblotting. Anti-His antibody was from Qiagen (Valencia, CA, USA), while β-actin was from Santa Cruz Biotech. (Santa Cruz, CA, USA). The blots were first probed with primary antibodies, followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized with the ECL system (Amersham).
RNA isolation and RNA-Seq
Hela were transfected with human Tip110 siRNA or the universal control siRNA. Total RNA was purified from transfected cells using Trizol according to the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA) and used for RNA-Seq. The RNA-Seq was performed at the Genomic Core of UT Southwestern Medical Center, Dallas, TX, USA. The RNA-Seq reads were aligned to the human genome sequences for further differential and spliceosome pathway analysis using the Panther Pathway Analysis software.
Total RNA was extracted using Trizol and used to synthesize cDNA using a ScriptII RT reagent kit (Promega). cDNAs were used for qPCR using a Power Sybr®Green PCR kit (Life Technologies) according to the manufacturer’s instructions. The qPCR program consisted of one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min.