Cloning and mutagenesis
A minigene construct containing the last 156 nucleotides (nt) of exon 4 (E4), I4 and the first 167 nt of exon 5 (E5) of Mus musculus fosB was created for use as a template for splicing and binding assays (a gift of Dr. Y. Nakabeppu, Kyushu University, Japan). The fosB minigene was amplified by PCR and subcloned into both the pGEM-T Easy plasmid (Promega, Madison, WI), adjacent to the T7 promoter to create pEZ_ISS/WT, and a modified pGL3 plasmid (Promega), adjacent to a SV40 promoter to create ISS/WT for transfection studies.
pEZ ISS/WT was used as a template to generate the mutant pEZ_ ISS/T6. A set of complementary oligonucleotides containing the desired mutations was synthesized () and used as primers for site-directed mutagenesis using a Quickchange kit (Stratagene, La Jolla, CA). Nucleotides 128 and 130 within the alternatively spliced intron were changed from C to T (C128T129C130→TTT), in order to convert a putative PTB-binding site into a pyrimidine-rich sequence not recognized by PTB. The mutated fosB minigene was amplified by PCR using 50 ng of pEZ_ISS/WT construct, 125 ng of sense and anti-sense primers, 0.2 mM each of dTTP, dATP, dCTP and dGTP and 2.5 U of PfuTurbo DNA polymerase (Stratagene) in a buffer containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4, 0.1% Triton X-100 and 0.1 mg/ml bovine serum albumin (BSA). Eight percent dimethylsulphoxide (DMSO) was added to the reaction to increase the specific binding of the primers. The cycling parameters used were as follows: denature at 95°C for 30 sec, 20–30 cycles at 95°C for 30 sec, 55°C for 1 min, and 68°C for 8 min. Reactions were cooled to 37°C and incubated with Dpn I (10 U/µl) for 1 hr in order to digest the parental template so that only mutated DNA remains. Constructs were sequenced to confirm that the mutation. The pEZ_ISS/T6 sequence was also subcloned into a modified pGL3 plasmid (Promega), adjacent to a SV40 promoter to create ISS/T6 for transfection studies.
Nuclear extract preparation, transcription and in vitro splicing assays
Soluble extracts were prepared from nuclei isolated from 11 liters of HeLa cells grown in spinner cultures using a modification of the Dignam method 
. Templates for splicing substrates were generated by digestion of pEZ_ISS/WT with Nar
I. Labeled RNA was synthesized from linear plasmid in a 50 µl reaction containing 2 µg of DNA, 0.4 mM each of ATP and GTP, 0.1 mM UTP and CTP, 2.5 µl of [32
P]-UTP and [32
P]-CTP (ICN), 0.5 mM diguanosine triphosphate and 2 µl of 20 U/µl T7 RNA polymerase Plus (Ambion, Austin, TX). Reactions were incubated at 37°C for 1 hr and then digested with DNase I for 15 min at 37°C. Transcripts were purified on 4% denaturing polyacrylamide gels, cut from the gel and eluted on a rotating platform in RNA elution buffer containing 2 M ammonium acetate, 1% SDS and 25 µg/ml yeast tRNA at 37°C overnight. RNA substrate was then extracted with phenol:chloroform and ethanol precipitated. In vitro
splicing assays were done as previously described using 11 µl of HeLa extract (2.6 µg/µl) in a total reaction of 25 µl 
. Spliced products were separated on 4% denaturing polyacrylamide gels.
Templates for intronic transcripts were prepared by PCR using oligonucleotides listed in . All forward primers contained a T7 RNA polymerase promoter. Each PCR reaction (20 µl) contained 0.88 ng of pEZ_ISS/WT template, 20 pmol of each primer, 0.25 µl Taq polymerase (5U/µl, Promega), 2 µl of 10× Taq buffer (Promega) and 1 µl of dNTP mix containing 10 mM dATP, dCTP, dGTP and dTTP. The parameters were as follows for 35 cycles: denaturation at 94°C for 15 sec, annealing for 15 sec at variable temperatures dependent upon the primers (5′ intron template primers: 53.3°C, middle intron template primers: 73.1°C, and 3′ intron template primers: 67.9°C), and extension: 72°C for 30 sec. PCR reactions were then extracted with phenol:chloroform and ethanol precipitated.
Intronic transcripts were prepared using PCR generated templates. Transcription reactions (50 µl) contained 2 µg of template, 2.5 µl of 200 U/µl T7 RNA polymerase Plus (Ambion), transcription buffer (Ambion), 16 mM MgCl2, 10 mM dithiothreitol (DTT), 4 mM each of ATP, CTP, GTP and UTP and 40 units of RNasin (Promega). Reactions were incubated at 37°C for 1–2 hr and then RQ DNAse (Promega) was added and incubated for an additional 15 min. RNA was phenol:chloroform extracted, ethanol precipitated and resuspended in water. RNA was quantified by measuring absorbance at 260 nm.
Radioactively-labeled intron transcripts were synthesized in 50 µl reactions containing 1 µg of PCR generated template, 0.5 mM each of ATP and GTP, 0.1 mM UTP and CTP, 2.5 µl of [32P]-UTP and [32P]-CTP (ICN) and 2 µl of 200 U/µl T7 RNA polymerase Plus (Ambion). Reactions were incubated at 37°C for 1 hr and then digested with DNase I for 15 min at 37°C. Transcripts were purified on 10% denaturing polyacrylamide gels and then processed as described for the splicing substrates.
Identification of fosB splicing products
Products of splicing reactions using transcripts generated from the fosB minigene constructs were cut out of the gel and eluted overnight in Elution Buffer. RNA was ethanol precipitated and reverse transcribed into cDNA (SuperScript II RT, Stratagene) in a 20 µl reaction containing 50 ng random primers, 5 µl of RNA and 1 µl dNTP mix (10 mM each). The mixture was heated to 65°C for 5 min and 4 µl of 5× first strand buffer, 2 µl of 0.1 M DTT and 1 µl RNaseOUT (40 units/µl) were added to the reaction. SuperScript II RT (1 µl, 200 U/µl, Invitrogen, Carlsbad, CA) was added to the reaction and incubated at 42°C for 50 minutes. The reaction was inactivated by incubation for 15 min at 70°C. PCR reactions (50 µl) contained 5 µl 10× PCR buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 µl 50 mM MgCl2, 10 mM dNTP mix, 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 0.4 µl Taq DNA polymerase (5U/µl) and 2 µl cDNA. When intron 4 spliced (IS) and intron 4 retained (IR) RNAs were amplified, a set of E4- and E5-specific primers were used (). An I4-specific forward primer and an E5-specific reverse primer were used to amplify lariat structures (). Samples underwent 35 cycles of amplification using the following cycling parameters: 15 sec at 94°C, 15 sec at 60°C or 66°C depending on the primers used, and 30 sec at 68°C. The PCR products were separated on 2% agarose gels.
UV cross-linking assays
UV cross-linking assays were done as previously described 
. Assays contained 50,000 cpm of [32
P]UTP labeled 5′, middle and 3′ substrates in a 10 µl reaction containing 5 µl of HeLa extract (2.6 µg/µl). UV cross-linking reactions were assembled on ice, incubated for 3 min at 30°C and then irradiated in open 1.5 ml centrifuge tubes with 1.2 J, 6.5 cm from a UV light source using a Stratalinker (Stratagene). After exposure to UV light, the reactions were incubated with RNase A (1 mg/ml, Sigma-Aldrich, Saint Louis, MO) for 20 min at 30°C. Reactions were terminated by the addition of protein loading buffer and heating at 95°C for 5 min before loading on 10% SDS polyacrylamide gels. The gels were fixed with 45% methanol and 9% acetic acid before drying and exposing to film.
UV cross-linking immunoprecipitations
P]UTP 5′, middle or 3′ RNA substrates were UV cross-linked and RNase A treated as described above. A 10 µl aliquot of a 1:50 dilution of the anti-PTB antibody, Bb7 
(a gift of Dr. D.L. Black, UCLA), anti-U2AF65
(Santa Cruz Biotechnology, Santa Cruz, CA) or nonspecific antibody (anti-RIF 2C1, a gift of Dr. K. Beaman, RFUMS) was added to each reaction and incubated at 4°C overnight. The following day 20 µl of Protein A agarose (Roche Diagnostics, Mannheim, Germany) was added to each reaction prior to incubation at room temperature for 1 hr. NET buffer (1 ml) containing 50 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.5% NP40 was added to each sample. Precipitates were collected by centrifugation at 600 × g at 4°C for 30 sec. The samples were washed four times in NET buffer. After the final wash the precipitate was resuspended in loading buffer, boiled for 3 min and then loaded on 10% SDS polyacrylamide gels. The gels were fixed with 45% methanol and 9% acetic acid before drying and exposing to film.
Cell Culture and Transient expression assays
HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum. His-tagged PTB 1 (human PTB 1, a gift from Dr. J. Patton, Vanderbilt University), was transiently transfected into HeLa cells plated at ~75% confluency in a 100 mm culture dish using the Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. Twenty four hours after transfection the cells were trypsinized and replated into two 100 mm culture dishes and cell lysates were collected the following day for protein isolation as described below. Slight modifications of the methods described above were also used to transiently transfect pBS (bluescript); ISS/WT; ISS/T6; and PTB 1 constructs into HeLa cells for RNA analysis. Cells were plated into 12 well culture plates (~35% confluency) ~4 hr prior to transfection. Each experimental condition was performed in duplicate. Additionally, each experimental protocol was performed at least three times. Forty-eight hr post-transfection RNA was isolated using the Trizol method, DNase-treated as described above, and cDNA generated. The cDNA was amplified by PCR using a forward primer designed to be complementary to sequence within the pGL3 vector, therefore, only ectopically expressed fosB sequence was amplified. The reverse primer was located in exon 5, thus allowing the amplification of both fosB and ΔfosB cDNA in a PCR reaction with a single set of primers. Control PCR reactions with primers specific for GAPDH were also performed. The amplified products were resolved on 2.5% agarose gels then imaged and quantitated using Kodak 1D Image Analysis Software (Kodak, Rochester, NY).
PTB protein isolation
The Ni-NTA Purification System (Invitrogen) was used for the purification of polyhistidine-containing recombinant proteins. Cells were suspended in 1.5 ml native binding buffer (250 mM NaH2
pH 8.0/2.5 M NaCl) supplemented with a protease inhibitor cocktail (Roche Diagnostics) and a phosphatase inhibitor cocktail (1
100, Sigma-Aldrich). Cells were lysed by a freeze-thaw cycle and the DNA sheared by passing the resulting cell lysate through an 18-gauge needle followed by centrifuged at 3,000×g for 15 min. Ni-NTA columns were prepared by washing 75 µl of Ni-NTA agarose three times with native binding buffer. Lysate (1.5 ml) was then incubated on the column for 30 min, followed by four washes with native wash buffer (native binding buffer supplemented with 20 mM imidazole, pH 8.0). The supernatant was discarded and protein was eluted with 150 µl native elution buffer (native binding buffer supplemented with 250 mM imidazole, pH 8.0). Extracts were loaded into Slide-A-Lyzer Mini Dialysis Units (10,000 MWCO, Pierce, Rockford, IL) and dialyzed overnight into Buffer D (20 mM HEPES-KOH pH 8.0, 20% glycerol, 100 mM KCl and 0.2 mM EDTA pH 7.9). Purified proteins were examined by Western blotting using PTB or His-tag specific antibodies.
Phosphatase treatment and PKA assay
Uniformly-labeled [32P]UTP and [32P]CTP fosB RNA substrates containing the 3′ end of I4 were UV cross-linked and RNase A treated as described above. Six microliters of purified His-tagged PTB (0.25 µg/µl) was used in the cross-linking assays. For phosphatase treatment, protein was pre-incubated for 15 min with NE Buffer 3 (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT) followed by a 50 min incubation at 37°C with 3 µl (10 U/µl) of calf intestinal alkaline phosphatase (CIP, New England Biolabs, Ipswitch, MA). For the PKA phosphorylation reaction, protein was incubated with 3 µl of the catalytic subunit of cAMP dependent protein kinase (New England Biolabs, 2,500 U/µl), 200 µM ATP, 1× PKA Reaction Buffer (50 mM Tris-HCl pH 7.5 and 10 mM MgCl2) in a total reaction volume of 30 µl, for 1 hr at 30°C. Some of the His-tagged protein samples were phosphatase and PKA treated. In these instances, the phosphatase step was performed while the protein was still on the nickel beads, followed by wash out of the phosphatase, elution of the His-tagged protein and subsequent PKA treatment.
Western blot analysis
Samples were mixed with an equal volume of 2× SDS sample buffer (125 mM TrisHCl pH 6.8, 20% glycerol, 4% SDS, 0.2% 2-mercaptoethanol (2-ME) and 0.001% bromphenol blue), boiled for 3 min and resolved by electrophoresis in an 8% SDS-polyacrylamide gel. Proteins were transferred to PVDF-Plus transfer membranes (Fisher Scientific, Pittsburgh, PA) for immunoblotting, using a semi-dry transfer apparatus (Owl Scientific, Woburn, MA) according to manufacturer's instructions.
PVDF membranes were then rinsed three times in 0.1% Tween-20 in TBS (TBS-T), blocked with 2% milk for 1 hr at room temperature, incubated with either PTB antibody (1/4,000 dilution; a gift of Dr. D.L. Black, UCLA) overnight at 4°C, washed with TBS-T and incubated for 60 min at room temperature with ECL peroxidase-labelled anti-mouse IgG (1/4,000; Amersham Biosciences). After three final washes in TBS-T, membranes were rinsed in TBS, immersed in chemiluminescence detection reagent (ECL Plus, Amersham Biosciences) and exposed to HyperFilm ECL film (Amersham Biosciences). Antibodies were stripped from the membrane by incubating in 62.5 mM TrisHCl (pH 6.8), 2% SDS, 100 mM 2-ME at 50°C for 30 min. Blots were then probed with an α-polyhistidine antibody (1/3,000 dilution; Clone His 1, Sigma-Aldrich). Images were captured using Kodak 1D Image Analysis Software.
Transcripts which have been shown to effectively deplete PTB from HeLa nuclear extracts 
were generated from a plasmid containing the CUCUU-octamer sequence (a gift of Dr. C.W. Smith, University of Cambridge). RNA transcription was carried out in the presence of 100 µM biotin-14-CTP as previously described 
. The biotinylated RNA was then bound to streptavidin magnetic beads (Invitrogen; 100 pmol of RNA/50 µl of beads) in 2× BW buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 2 M NaCl). One hundred microliters of HeLa nuclear extract (2.6 µg/µl) was preincubated with 0.5 µl of 100 mM DTT and 34 U of RNasin for 15 min at room temperature, followed by incubation with the RNA-streptavidin beads for 30 min, using 1,440 fmol of RNA/µl of extract. The beads were removed from the extract using a magnet as described by the manufacturer.