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J. Biol. Chem., Vol. 282, Issue 33, 24444-24454, August 17, 2007

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Nuclear Protein TIA-1 Regulates COL2A1 Alternative Splicing and Interacts with Precursor mRNA and Genomic DNA^*

Audrey McAlinden¹, Li Liang, Yoshiki Mukudai, Toshihiro Imamura², and Linda J. Sandell

From the Departments of Orthopaedic Surgery and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, March 29, 2007 , and in revised form, June 5, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The RNA-binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions in regulating post-transcriptional mechanisms, including precursor mRNA (pre-mRNA) alternative splicing and mRNA translation. Utilizing a mini-gene consisting of part of the type II procollagen gene (COL2A1), we show that TIA-1 interacts with a conserved AU-rich cis element in COL2A1 intron 2 and modulates alternative splicing of exon 2. This unique, highly conserved cis element containing stem-loop secondary structure was previously identified in our laboratory as an essential motif that controls the developmentally regulated exon 2 splicing switch during chondrogenesis (McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711). In vivo binding of endogenous TIA-1 to the AU-rich cis element in COL2A1 pre-mRNA was confirmed by the ribonucleoprotein immunoprecipitation assay. Importantly, we also show that TIA-1 interacts with the equivalent DNA sequence with a preference for single-stranded rather than double-stranded DNA. Chromatin immunoprecipitation assays (including an additional RNase step) confirmed this interaction in vivo. Competition assays showed that TIA-1 apparently binds with higher affinity to DNA than to RNA. Finally, we show that this strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. This suggests a potentially novel, dual role for TIA-1 in shuttling between DNA and RNA ligands to co-regulate COL2A1 expression at the level of transcription and pre-mRNA alternative splicing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Gene expression is regulated on many levels from transcriptional activation and repression of genomic DNA to post-transcriptional mechanisms that control precursor mRNA (pre-mRNA)³ splicing and maturation as well as mRNA transport, turnover, and translation. In general, these mechanisms are regulated by interactions of DNA/RNA cis elements with specific trans-acting proteins, and numerous studies suggest that many processes of transcription and post-transcription are co-regulated in the nucleus (1).

With respect to pre-mRNA regulation, RNA-binding proteins are known to assemble on the message at the time of transcription, thus determining the fate of the transcript from the very beginning of gene activation (2, 3). Of these RNA-binding proteins, TIA-1 (T-cell-restricted intracellular antigen-1) is a well described regulator of mRNA metabolism in the cell. TIA-1 belongs to the RNA recognition motif (RRM)/ribonucleoprotein family of RNA-binding proteins (4, 5). Interactions of TIA-1 with RNA are mediated by the second of its three RRM domains (6), whereas RRM2 and RRM3 are both required for nucleocytoplasmic shuttling (7). Recently, microarray and computational analyses revealed that common TIA-1 binding motifs in mRNA consist of U-rich stretches that have the potential to form stem-loop secondary structures (8).

In the nucleus, TIA-1 has been shown to regulate pre-mRNA alternative splicing of genes such as those encoding fibroblast growth factor receptor-2 (FGFR-2), Fas receptor, cystic fibrosis transmembrane conductance regulator (CFTR), TIA-1-related protein (TIAR), and even TIA-1 itself (9-12). In these genes, the alternatively spliced exons contain weak 5' splice site sequences that do not conform to the bona fide consensus sequence (13). Consequently, TIA-1 functions to strengthen these weak splice sites by interacting with U-rich sequences downstream of the alternative exon. In doing so, TIA-1 can then facilitate recruitment of the U1 small nuclear ribonucleoprotein complex to the spliceosome to promote inclusion (splicing) of the alternative exon in the final mRNA transcript (14). Based on these observations of TIA-1 binding sites and function, we hypothesized that TIA-1 may also regulate pre-mRNA splicing of the type II procollagen gene (COL2A1).

COL2A1 encodes the major collagenous protein present in cartilage tissue (15), and its synthesis is developmentally regulated during chondrogenesis (16, 17). Chondro-progenitor cells synthesize the type IIA procollagen mRNA isoform containing alternative exon 2, whereas differentiated chondrocytes synthesize the type IIB mRNA isoform, devoid of exon 2 (18). This is one of the most important splicing events identified in connective tissue development and is likely a key mechanism for the production and maintenance of healthy cartilage. Recently, we showed that (i) COL2A1 exon 2 contains a weak 5' splice site sequence, (ii) an AU-rich cis element is present directly adjacent to the 5' splice site of exon 2 and is required for correct alternative splicing of COL2A1 exon 2, (iii) this RNA cis element contains secondary structure in the form of a stem-loop, and (iv) the AU-rich sequence within the double-stranded stem region is 100% conserved between species (19). In addition, this cis element contains repeats of the pentameric sequence AUUUA; these AU-rich elements (AREs) are commonly found in 3'-UTRs of mRNA transcripts thereby regulating their stability and translation efficiency (20, 21). In this respect, a cytosolic function has also been reported for TIA-1 through its interaction with AUUUA-containing cis elements in the 3'-untranslated region (UTR) of mRNAs encoding tumor necrosis factor-, cyclooxygenase-2, and cytochrome c and, in doing so, suppresses their translation (22-25).

In the present study, we show that TIA-1 enhances splicing of COL2A1 exon 2 and that this function is dependent on interaction with the conserved AU-rich cis element in intron 2. Of particular interest is our finding that TIA-1 can also bind to the equivalent AT-rich element in COL2A1 genomic DNA. We also show that the strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. We therefore propose a dual role for TIA-1 in co-regulating processes of transcription and pre-mRNA alternative splicing to control expression patterns of COL2A1 by potentially shuttling between DNA and RNA ligands.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of Wild-type and Mutant COL2A1 Mini-genes—A wild-type human COL2A1 mini-gene was constructed as previously described (19). This mini-gene (5.9kb) consists of exons 1-3 and full-length introns 1 and 2 (Fig. 1A). Mutant mini-genes were generated using the QuikChange^TM site-directed mutagenesis kit (Stratagene). Here, complementary primer pairs were designed to either delete all or a portion of the conserved stem-loop cis element previously identified in our laboratory (19) (Fig. 1B), whereas other mutant mini-genes were synthesized containing point mutations within the U-rich region of the stem-loop (Fig. 2). PCR mutagenesis was carried out over 18 cycles (95 °C, 30 s; 55 °C, 1 min; and 68 °C, 6 min) and resulting PCR products were treated with DpnI (1 µl) for 1 h at 37 °C to digest parental, methylated DNA. An aliquot (1 µl) of digested DNA was transformed into XL-1 Blue Super-competent Cells (Stratagene), and resulting colonies were screened for the presence of the correct mutation. For use in transfection assays, wild-type and all mutant mini-genes were cloned into pcDNA3 vector (Invitrogen) under transcriptional control of the cytomegalovirus promoter.

Transient Co-transfection of COL2A1 Mini-genes with TIA-1 and Analysis of Mini-gene Spliced Isoforms—In all studies, TC28/I2 chondrocytes from human juvenile costal cartilage were used (a gift from Dr. Mary Goldring, Cornell University). Constructs encoding either wild-type or mutant COL2A1 mini-genes were co-transfected with a TIA-1-expressing construct (from Dr. Jane Wu, Northwestern University) into these TC28 cells. Each mini-gene construct (1 µg) together with the TIA-1 construct (5 µg) were co-transfected using FuGENE^TM6 reagent (Roche Applied Science) according to the manufacturer's instructions. The first5hof transfection was carried out in serum-free medium, and, thereafter, cells were cultured in medium containing 10% serum for a further 48 h until RNA isolation. Total RNA was harvested and purified using the RNeasy kit (Qiagen), and an aliquot (1 µg) was reverse-transcribed using random primers and SuperScript^TM II RNase H-reverse transcriptase (Invitrogen). Resulting cDNA was used for quantitative PCR in the presence of [-³²P]dCTP (3000 Ci/mmol, Amersham Biosciences). The primers pcDNA3-COL2A1-Ex1 (5'-CAAGCTTACATGATTCGGC-3') and sp6 were used to amplify cDNA derived only from the exogenously transfected COL2A1 mini-gene. The linear range for these primers was established, and PCR was carried out for 20 cycles (95 °C, 30 s; 55 °C, 30 s; and 72 °C, 30 s). 10 µl of 6x loading dye (30% glycerol, 0.025% (w/v) bromphenol blue, 0.025% (w/v) Cyanol Blue) was added to each reaction, and 7 µl was electrophoresed at 200 V through 6% polyacrylamide gels. Gels were dried and exposed to a phosphorimaging screen for 1 h and then scanned on a STORM^TM 840 PhosphorImager (Amersham Biosciences). Bands corresponding to the type IIA (386 bp) and type IIB (179 bp) mRNA isoforms were quantified whenever necessary using ImageQuant^TM software.

Detection of Endogenous TIA-1 mRNA and Protein—Total RNA was extracted and purified from TC28 cells using the RNeasy kit (Qiagen). PCR was carried out on reverse-transcribed cDNA using primer pairs specific for human (NM_022173) TIA-1. Sense, forward primer corresponding to a sequence in exon 1 was: 5'-ATGGAGGACGAGATGCCCAA-3'. Reverse primer designed in exon 6 was: 5'-TGTGGGCTGAGATCACCA-3'. PCR was carried out in the presence of [-³²P]dCTP (3000 Ci/mmol, Amersham Biosciences). TIA-1 or -actin was amplified in the linear range established for each primer pair (27x cycles for TIA-1 or 20x cycles for -actin) using the same PCR parameters (95 °C, 30 s; 55 °C, 30 s; and 72 °C, 30 s). Radioactive cDNA bands were analyzed by gel electrophoresis and phosphorimaging as described above. Nuclear proteins from 2-4 x 10⁷ TC28 cells were extracted using the CelLytic^TM NuCLEAR^TM extraction kit (Sigma). Approximately 60-100 µg of nuclear extract was electrophoresed through 4-20% SDS-polyacrylamide gels followed by Western blotting onto a Hybond^TM-C membrane (Amersham Biosciences). TIA-1 was localized by a monoclonal antibody (ML-29, gift from the Dr. Paul Anderson laboratory, Harvard University) or polyclonal antibody (SC-1751, Santa Cruz Biotechnology, Santa Cruz, CA). Detection of horseradish peroxidase-conjugated secondary antibodies bound to localized TIA-1 antibodies was carried out using the ECL plus Western blotting detection system kit (Amersham Biosciences).

Synthesis of GST/TIA-1 Fusion Protein—Full-length human TIA-1 cDNA ("variant 2" containing alternative exon 5; NM_022173) was amplified from TC28 human chondrocytes using the primers: H-TIA-1-F-BamHI (5'-TAAGGATCCATGGAGGACGAGATGCCCAAG-3') and H-TIA-1-R-EcoRI (5'-TAAGAATTCTCACTGGGTTTCATACC-3'). TIA-1 cDNA was cloned into pGEX4T-1 vector (Amersham Biosciences) containing cDNA encoding glutathione S-transferase (GST) upstream of the multiple cloning site. Resulting GST/TIA-1-pGEX4T-1 fusion construct was transformed into BL21 cells (Amersham Biosciences). Overnight cultures of transformed cells were diluted 1:400 in LB media and grown at 37 °C until an A₆₀₀ of 0.6 was reached. Isopropyl 1-thio--D-galactopyranoside was then added to a final concentration of 0.1 mM, and growth was continued until reaching an A₆₀₀ of 1.4. Cells were collected, resuspended in cold 1x phosphate-buffered saline containing 0.5 M EDTA, and sonicated 5x for 30 s. Soluble protein in the supernatant was collected by centrifugation, mixed with pre-washed glutathione-Sepharose 4B (50% slurry) for 1 h at 4 °C and washed 3x with cold 1x phosphate-buffered saline. The protein was eluted with GEB (10 mM glutathione reductase in 50 mM Tris-HCl, pH 8.0). GST/TIA-1 protein was electrophoresed through a 4-20% polyacrylamide gel and either stained with Coomassie Blue or transferred to Hybond^TM-C membrane (Amersham Biosciences) for Western blotting. Membranes were then probed with either a TIA-1 antibody (monoclonal antibody ML29 from Dr. Paul Anderson's laboratory or a polyclonal antibody from Santa Cruz Biotechnology) or an anti-GST antibody to confirm the molecular weight and purity of the fusion protein.

RNA Electrophoretic Mobility Shift Assay—To construct full-length "stem-loop" 38-nucleotide sense RNA radiolabeled probes, antisense DNA oligonucleotides were first synthesized (Invitrogen) containing a 3' T7 leader sequence. The following DNA oligonucleotide was designed to synthesize the wild-type sense RNA probe: 5'-GTAATTTATTTATGTTGAACAGGAAATAAATAAATTACCCTGTCTC-3'. To synthesize the CCC-3 mutant RNA probe, the following DNA oligonucleotide was made: 5'-GTAATTTATTTATGTTGAACAGGGGGTGGGTGGGTTACCCTGTCTC-3'. In each oligonucleotide, the T7 leader sequence is shown in bold, underlined font. A control RNA probe was also synthesized, which corresponds to a 68-nucleotide probe previously shown to bind to TIA-1 (RNA sequence "1-6" from Dember et al.) (6).

The mirVana microRNA probe construction kit (Ambion) was used according to the manufacturer's instructions to synthesize sense RNA radiolabeled probes. Briefly, DNA oligonucleotide template (200 µM) was hybridized with the T7 promoter primer by incubation at 70 °C for 5 min followed by 5-min incubation at room temperature in DNA hybridization buffer. A double-stranded DNA template was generated by addition of Klenow DNA polymerase for 30 min at 37 °C. Transcription was carried out in the presence of T7 RNA polymerase and 50 µCi of [-³²P]UTP (3000 Ci/mmol) for 30 min at 37 °C, and template DNA was removed by addition of DNase I for 10 min at 37 °C. GST or GST/TIA-1 recombinant protein was added to 5 x 10⁴ cpm of radiolabeled RNA probe in RNA binding buffer (5 mM HEPES, pH 7.9, 7.5 mM KCl, 0.5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1 mg/ml yeast tRNA, and 0.1 mg/ml bovine serum albumin) for 30 min at 25 °C. The protein/probe binding mixture was then incubated with 1 µl of 1/100 diluted RNase mixture (0.5 unit/µl of RNase A and 20 units/µl RNase T1; Ambion) for a further 10 min at 37 °C. RNA·protein complexes were electrophoresed through 6% native polyacrylamide gels in 0.5x TBE buffer (40 mM Tris-HCl, 40 mM boric acid, 1 mM EDTA, pH 8.3) and visualized by autoradiography. For competition assays, the recombinant protein was added to a mixture of RNA radiolabeled probe and either DNA or RNA cold competitor probe and incubated at 25 °C for 30 min before gel electrophoresis.

UV Cross-linking—RNA-protein binding reactions were carried out as described in the previous subsection. Following the RNase digestion step, RNA·protein complexes were placed on ice until UV irradiation in a CL-1000 UV cross-linker (UVP) for 10 min. Samples were then heated for 5 min in SDS protein gel sample buffer and separated by 4-20% SDS-PAGE. Gels were then dried, and cross-linked complexes were viewed by autoradiography.

Ribonucleoprotein Immunoprecipitation Assay—Analysis of endogenous TIA-1 interaction with the COL2A1 pre-mRNA region of interest (Fig. 6) was done according to previously described protocols (8, 26). Briefly, protein G-Sepharose beads (Sigma) were pre-swollen in NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40) supplemented with 5% bovine serum albumin. Beads (100 µl) were incubated for 16 h at 4 °C overnight with either TIA-1 antibody (20 µg, Santa Cruz Biotechnology) or normal goat IgG (20 µg, Santa Cruz Biotechnology). Antibody-coated beads (50 µl) were then added to 100-µl cell lysates (extracted from 14 x 10⁶ cultured TC28 cells) for 2 h at 4 °C with constant mixing. After extensive washes, DNA and protein in the immunoprecipitation (IP) reaction was digested with DNase I for 10 min at 37 °C and then proteinase K for 30 min at 55 °C. RNA was eluted from the beads and purified by phenol-chloroform extraction. Reverse transcription-PCR was then carried out using the primer pair to amplify the region of interest in intron 2 of COL2A1 (Fig. 5): sense primer, 5'-CTGGACCTCGCCACTGCCAGTG-3'; antisense primer, 5'-TGATCTCCATAGTGGCTCTAG-3'. Reverse-transcribed RNA was extracted and immunoprecipitated from TC28 cells that had been transfected with the COL2A1 mini-gene for 48 h. This was done to provide enough COL2A1 pre-mRNA substrate as the normal rate of active COL2A1 transcription in these cells is very low.⁴ PCR was then carried out (95 °C, 1 min; 55 °C, 30 s; and 72 °C, 30 s) for 30 cycles, and cDNA products were visualized after electrophoresis through ethidium bromide-containing 1% agarose gels. PCR without a prior reverse transcription step was done to ensure there was no DNA in the immunoprecipitated sample.

DNA Electrophoretic Mobility Shift Assay—Sense and complementary DNA oligonucleotides, corresponding to the wild-type 38-nucleotide RNA stem-loop cis element, were synthesized by Invitrogen. For double-stranded probes, sense and antisense oligonucleotides were annealed in TSE buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0) by heating to 95 °C and slowly cooling to room temperature. All double-stranded or single-stranded oligonucleotides were end-labeled using T4 polynucleotide kinase and [-³²P]ATP. Radiolabeled single-stranded or double-stranded DNA probes (1 x 10⁴ cpm) were incubated with various concentrations of purified GST or GST/TIA-1 fusion protein in the presence of 3 µl of 5x binding buffer (25% glycerol, 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM MgCl₂, 0.5 mM EDTA, 5 mM dithiothreitol), 3 µg of poly(dI-dC) in a total volume of 15 µl. Binding reactions were incubated for 30 min at room temperature, and protein/probes were electrophoresed through 4% polyacrylamide gels in 0.5x Tris borate-EDTA buffer. Gels were dried, exposed to a phosphorimaging screen, and scanned on a STORM^TM 840 PhosphorImager (Amersham Biosciences). For DNA competitor binding assays, known quantities of unlabeled competitor probe was mixed with recombinant protein on ice for 30 min followed by addition of the radiolabeled DNA probe for 30 min at room temperature. For RNA competitor assays, specific amounts of unlabeled RNA probe were added to labeled DNA probe followed by addition of recombinant protein for 30 min at room temperature.

ChIP Assay—Chromatin immunoprecipitation (ChIP) assays were carried out using the EZ ChIP^TM assay kit (Upstate Biotechnology) according to the manufacturer's instructions. TC28 cells were used because they contain abundant endogenous TIA-1 protein, and overexpression of TIA-1 in these cells altered the splicing pattern of COL2A1 (Fig. 2). One 15-cm dish of TC28 cells (4 x 10⁶) in 15-cm culture dishes was used for one IP reaction. Cross-linking of DNA·proteins was induced by addition of formaldehyde (1% final concentration) directly to the culture medium for 10 min at 37 °C. Cells were lysed and DNA in the supernatant was sheared by sonication. An aliquot of the sonicated chromatin sample ("input") was removed for PCR analysis, and the remainder was used for IPs. Prior to addition of antibody, some samples were treated with RNase A/T1 mixture (final concentration of 7.5 units of RNase A, 300 units of RNase T1) (Ambion) or an equivalent volume of RNase storage buffer (10 mM Hepes, pH 7.2, 20 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, and 50% glycerol, v/v) for 30 min at room temperature to digest any native RNA transcripts present in the chromatin complex. To each RNase with or without supernatant sample was added either 5 µg of TIA-1 antibody (Santa Cruz Biotechnology) or normal goat IgG (Santa Cruz Biotechnology) that had been pre-coated onto Protein G-Sepharose beads, and reactions were incubated overnight with constant mixing at 4 °C. Protein G beads were collected by centrifugation and protein·DNA complexes were eluted from the beads followed by a cross-link reversal step by addition of 5 M NaCl (8 µl) to the eluted sample (200 µl) for 4 h at 65 °C.DNA from each IP reaction (2 µl) was used for PCR with a primer pair to amplify the 355-bp region of interest in COL2A1 intron 2 (Fig. 7): sense primer, 5'-CTGGACCTCGCCACTGCCAGTG-3'; antisense primer, 5'-TGATCTCCATAGTGGCTCTAG-3'. A second set of primers were designed to amplify a 222-bp region further downstream in COL2A1 intron 2 that we predicted would not contain a TIA-1 binding site (Primers 3 and 4; Fig. 7): sense primer, 5'-CCTCTCTGAAATCATCTCTG-3'; antisense primer, 5'-CTACCACCAATGTTCCATAAC-3'. Input or immunoprecipitated DNA was amplified by PCR (94 °C, 20 s; 59 °C, 30 s; and 72 °C, 2 min) for 29 cycles.

Transcription Displacement Assay—The RNA transcription assay was carried out as described previously (27). The following two oligonucleotides were synthesized (Invitrogen): T7, 5'-TAATACGACTCACTATAGGG-3' and T7-COL2A1-T/A, 5'-CATTAAATAAATACAACTTGTTCCTTTATTTATTTAATGCACTGGAATTCACCATACTATCCCTATAGTGAGTCGTATTA-3'. The latter sequence consists of the 38-nucleotide AT-rich cis element of COL2A1 (in bold font), 21 nucleotides of random sequence, and a complementary T7 sequence. The T7 and T7-COL2A1-T/A oligonucleotides were mixed in annealing buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA), boiled, and cooled to room temperature to form a duplex oligonucleotide in the T7 recognition sequence. This partially duplexed oligonucleotide (1.3 pmol) was used as a template in a T7 transcription reaction. Transcription was carried out using a kit (Ambion) in the presence of 5 µCi of [-³²P]UTP (3000 Ci/mmol) either with or without the addition of rCTP. Resulting radiolabeled RNA oligonucleotides were electrophoresed through an 8 M urea/6% polyacrylamide gel and visualized by autoradiography. For TIA-1 binding and displacement assays, the T7-COL2A1-T/A oligonucleotide (1.3 pmol) was end-labeled using T4 polynucleotide kinase and 50 µCi of [-³²P]ATP (3000 Ci/mmol) and then partially duplexed to the T7 oligonucleotide. TIA-1/GST fusion protein (300 ng) was added to the radiolabeled, duplexed oligonucleotide in RNA binding buffer (5 mM HEPES, pH 7.9, 7.5 mM KCl, 0.5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1 mg/ml yeast tRNA, and 0.1 mg/ml bovine serum albumin) for 30 min at 25 °C. The protein·oligonucleotide complex was added to the transcription reaction mix containing unlabeled UTP in the presence or absence of rCTP. T7 RNA polymerase (Ambion) was then added, and the reaction was incubated at 37 °C for various time intervals. Excess DNA was digested by DNase I (Invitrogen) followed by UV cross-linking as described previously. Resulting protein·oligonucleotide complexes were subjected to 4-20% SDS-PAGE and visualized by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TIA-1 Regulation of COL2A1 Alternative Splicing Is Dependent on the U-rich Region of the Stem-loop cis Element—Fig. 1A shows the structure of the type II procollagen (COL2A1) mini-gene used to study regulation of exon 2 alternative splicing. The two modes of alternative splicing to produce either the type IIA (+exon 2) or type IIB (-exon 2) mRNA isoforms derived from the mini-gene are shown, in addition to the expected sizes of each product after reverse transcription-PCR amplification. Fig. 1B shows the location of the RNA stem-loop cis element containing a highly conserved AU-rich sequence. This cis element was previously identified in our laboratory as an essential factor required for correct regulation of exon 2 splicing and was shown to contain RNA secondary structure (19). We then investigated the effect of TIA-1 on splicing of exon 2 given previous reports that this intracellular protein can regulate alternative splicing of other genes by binding to intronic U-rich elements located downstream of an alternatively spliced exon.

Fig. 2 shows that in human chondrocyte (TC28) cells, TIA-1 overexpression altered the splicing pattern of COL2A1, favoring exon 2 inclusion (splicing), i.e. production of the type IIA isoform. Here, the ratio of IIA:IIB spliced products increased 3-4 fold as measured by quantitative densitometry. We then investigated whether the alternative splicing function of TIA-1 in the TC28 chondrocytes is dependent on the AU-rich stem-loop element. A series of mutant mini-genes were constructed that deleted all (SL-Del 1) or part (SL-Del 2) of the stem-loop or contained point mutations within the U-rich region (CCC-1, CCC-2, and CCC-3). Fig. 2 shows that deleting all 38 nucleotides of the stem-loop or deleting the 12-nucleotide U-rich stretch (AUUUAUUUAUUU) inhibited splicing of exon 2, as shown previously (19). However, overexpression of TIA-1 with these mutant mini-genes inhibited the ability of TIA-1 to enhance exon 2 splicing (inclusion) to favor the type IIA isoform. TIA-1 did increase exon 2 splicing of the COL2A1 mutant mini-genes that contained UC substitutions in the first and second U triplets (CCC-1 and CCC-2). However, the splicing enhancing effect of TIA-1 was diminished when co-transfected with the CCC-3 mutant, suggesting that all three U-triplets are required for maximum TIA-1 function in regulating splicing of COL2A1 exon 2.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Alternative splicing of the COL2A1 mini-gene. The human COL2A1 mini-gene consists of exons (E) 1-3 and intervening introns 1 and 2 (A). Synthesis and cloning of this mini-gene has previously been described (19). P1 and P2 show the location of primers used to amplify the type IIA (+exon 2) or type IIB (-exon 2) mRNA transcripts derived from the mini-gene. Sizes of resulting IIA or IIB mRNA are also shown. Location and sequence of the conserved cis element required for differential splicing of COL2A1 exon 2 (B). The cis element within the precursor mRNA (denoted by the asterisk) is located between nucleotides +4 and +41 of intron 2. This stretch of pre-mRNA contains an AU-rich region that is conserved between species. RNA mapping studies showed that this cis element contains RNA secondary structure in the form of a stem-loop (19).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Effect of TIA-1 on alternative splicing of COL2A1 exon 2. TC28 chondrocytes were co-transfected with constructs expressing the human wild-type (WT) or mutant COL2A1 mini-genes and human TIA-1 protein or empty pcDNA3 vector (mini-gene: TIA-1/pcDNA3 ratio of 1:5). Boxed gel picture (top left) is a Western blot showing increased levels of TIA-1 protein in the nuclei of TC28 cells co-transfected with a TIA-1 cDNA construct (TIA-1) compared with non-transfected (NT) or mock-transfected (pcDNA3 only) cells. Polyclonal antibody from Santa Cruz Biotechnology was used to detect TIA-1 protein. Mutant mini-genes with deletions in the stem-loop cis element (SL-Del 1 and SL-Del 2) or containing point mutations in the U-rich stem-loop region (CCC-1, CCC-2, and CCC-3) were also co-transfected with a TIA-1-expressing construct (+) or empty pcDNA3 vector (-). Levels of IIA (+exon 2) and IIB (-exon 2) mRNA derived from either WT or mutant COL2A1 mini-genes were monitored by phosphorimaging and radioactive bands were quantified by ImageQuaNTTM software. All co-transfections were carried out a minimum of three times. The phosphorimaging results shown are representative of all experiments (minimum of three replicates).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Probes and recombinant proteins for in vitro binding assays. Sequence of wild-type and CCC-3 RNA or DNA probes. The T7 leader sequence in the RNA probes is shown in italics. Point mutations in the CCC-3 probes are underlined in bold font (A). Secondary structure of wild-type and CCC-3 RNA probes. Mfold software was used to show predicted secondary structure of the wild-type and CCC-3 RNA probes. Wild-type probe is shown to form a stem-loop structure (also proven experimentally (19)), whereas secondary structure is completely disrupted by introducing point mutations within the double-stranded stem region (B). Purified GST/TIA-1 and GST recombinant proteins. Approximately 3 µg of purified recombinant protein was electrophoresed through 4-20% SDS-polyacrylamide gels and stained with GelCode® Blue (Pierce). Molecular weight protein markers are shown in the left panel (C).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. In vitro binding of AU-rich RNA cis element to TIA-1. Phosphorimaging results of RNA·protein complexes after exposure to UV cross-linking and electrophoresis through a 4-20% SDS-PAGE gel. Wild-type (WT), CCC-3 mutant or positive control (CTL) RNA probes were incubated with increasing amounts of GST/TIA-1 fusion protein (200, 400, and 800 ng). Positive control (CTL) probe corresponds to a 68 nucleotide U-rich probe "1-6" previously shown to bind to TIA-1 (6) (A). Competition of TIA-1-RNA interactions (5 x 104 cpm probe + 600 ng of GST/TIA-1) with increasing amounts (50, 100, and 200 ng) of RNA competitor probes: wild-type (WT) RNA, CCC-3 RNA mutant and control (CTL) RNA 68-nucleotide probe. Addition of wild-type DNA single-stranded competitor probe (100, 200, and 400 ng) was also analyzed (B). The sequence of COL2A1 WT and mutant RNA and DNA probes is shown in Fig. 3A.

TIA-1 Interacts with COL2A1 Genomic DNA—Given the fact that DNA oligonucleotide probe could strongly compete with TIA-1 binding to the wild-type RNA probe (Fig. 4B), we further investigated the potential of a direct interaction between TIA-1 and COL2A1 genomic DNA. We synthesized the AT-rich radiolabeled DNA probe (Fig. 3A) corresponding to the 38-nucleotide cis element that we identified in intron 2 of COL2A1 pre-mRNA. Electrophoretic mobility shift assays were carried out to detect whether TIA-1 could interact with either the single-stranded or double-stranded DNA oligonucleotide. Fig. 6A confirms a direct interaction of TIA-1 to the DNA probe with an apparent preference for single-stranded DNA. The single-stranded free probe migrated at a faster rate through the gel than the double-stranded free probe. This suggests that, like the wild-type RNA oligonucleotide, a compact, secondary structure may exist within the single-stranded DNA probe that would likely be disrupted in the DNA double-stranded probe to form a more linear, unfolded structure. One major radiolabeled DNA·protein complex band was seen with addition of the highest amount (450 ng) of GST/TIA-1 protein to the single-stranded DNA probe. In the presence of less protein, two or three shifted bands were detected that, compared with the major shifted band, migrated to a different position in the gel. This pattern was also seen by addition of high levels of protein to the double-stranded DNA probe, therefore suggesting that this altered migration pattern may represent weaker/less efficient binding. Additional gel shift and competition assays confirmed that the T-rich region of the cis element was important for DNA binding, although some weaker binding of TIA-1 to the CCC-3 mutant DNA probe was observed (Fig. 6B).

An in vivo interaction between the COL2A1 genomic DNA intronic cis element and endogenous TIA-1 protein in the nucleus of TC28 cells was confirmed by ChIP assays (Fig. 7). The first two lanes of amplified PCR products show the regions of COL2A1 intron 2 amplified by either primer pairs "1 + 2" (355 bp spanning the AT-rich TIA-1 binding site) or "3 + 4" (222 bp region not expected to contain a TIA-1 binding site) before immunoprecipitation (input chromatin). After immunoprecipitation with TIA-1 antibody, the 355-bp band spanning the AT-rich TIA-1 binding site was detected: this cDNA fragment was present in the TIA-1 immunoprecipitated chromatin complex before and after RNase treatment, thereby confirming an interaction with genomic DNA and not with native RNA transcripts that are also likely to be present in the chromatin complex. There was a 4-fold minimum increase in the intensity of the 355-bp cDNA band that was amplified after TIA-1 IP compared with that amplified after IgG IP. There was no sign of endogenous TIA-1 binding to a random downstream region (222 bp) of COL2A1 intron 2 genomic DNA. Results shown are representative of three independent experiments.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. In vivo interaction of endogenous TIA-1 with COL2A1 pre-mRNA. TC28 human chondrocytes were transfected with the COL2A1 mini-gene and the ribonucleoprotein immunoprecipitation assay was carried out using an anti TIA-1 antibody or IgG antibody as a control. DNase I-treated RNA was reverse transcribed and amplified by PCR (30x cycles) using primers to specifically amplify the 355-bp region of COL2A1 pre-mRNA containing the AU-rich cis element of interest. A PCR control reaction in the absence of a prior reverse transcription step was also carried out.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. Interaction of TIA-1 with COL2A1 genomic DNA in vitro. Electrophoretic mobility shift assay phosphorimaging showing TIA-1·DNA complex formation. Single-stranded (ss) or double-stranded (ds) DNA probes (1 x 104 cpm) corresponding to the wild-type RNA stem-loop sequence was incubated with increasing concentrations (50, 150, or 450 ng) of GST or GST/TIA-1 recombinant proteins. X denotes probe alone without addition of protein (A). Competition of GST/TIA-1 (450 ng) binding to the single-stranded WT DNA probe (1 x 104 cpm) by increasing amounts of WT or CCC-3 unlabeled probes (ranging from 50 to 150 ng). Arrows denote the position of the major radiolabeled shifted complexes. X denotes probe-protein complex without addition of competitor probe (B).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. In vivo interaction of TIA-1 with COL2A1 genomic DNA. Top panel shows the two regions of COL2A1 genomic DNA that were amplified by primer pairs P1 + P2 (a 355-bp fragment spanning the potential AT-rich TIA-1 binding site) and P3 + P4 (a downstream 222-bp fragment not predicted to contain a TIA-1 binding site). Immunoprecipitations were carried out using chromatin complexes isolated from human TC28 chondrocyte cells. PCR was used to amplify either the 355-bp or 222-bp region of COL2A1 genomic DNA after immunoprecipitation with an anti-TIA-1 or IgG antibody. Chromatin samples that were pre-treated with RNase before immunoprecipitation are shown.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. Disruption of TIA-1-DNA interaction by RNA polymerase during transcription. Phosphorimaging scan of DNA-TIA-1-radiolabeled shifted complexes (450 ng of GST/TIA-1 + 1 x 104 cpm WT DNA probe) in the absence of competitor probe (-) or in the presence of the WT DNA or RNA full-length competitor probe (80 ng) (A). Oligonucleotide (T7-COL2A1-T/A) was used as a template for T7 RNA polymerase. This template was partially duplexed at the T7 promoter site as shown. The asterisk denotes the site where T7 RNA polymerase would stall in the absence of rCTP in the transcription reaction. Sequence in bold font represents COL2A1 genomic DNA cis element containing the TIA-1 binding site (B). In vitro transcription reaction using the partially duplexed T7-COL2A1-T/A oligonucleotide in the presence (lane 1) or absence (lane 2) of rCTP. RNA markers (M) are shown in the left lane panel (C). TIA-1 recombinant protein was bound to the partially duplexed T7-COL2A1-T/A radiolabeled oligonucleotide and incubated with T7 RNA polymerase in the presence of rNTPs for various time intervals from 0 to 180 min. DNA·TIA-1 complex was then subjected to UV cross-linking, electrophoresed by SDS-PAGE and visualized by autoradiography. Autoradiograph shows disruption in the TIA-1·DNA complex as the transcription reaction progresses over time. C1 denotes a control reaction in the absence of T7 RNA polymerase, and C2 represents a second control where rCTP was not added to the transcription reaction (D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

With respect to the function of TIA-1 in regulating pre-mRNA alternative splicing of COL2A1, we have shown that this RNA-binding protein is an enhancer splicing factor protein in human chondrocytes derived from juvenile costal cartilage (TC28 cells). Here, co-transfection of TIA-1 with the COL2A1 mini-gene induced exon 2 splicing (inclusion) favoring production of the chondro-progenitor type IIA isoform. It will be interesting to study if expression levels of TIA-1 (in addition to other trans-acting splicing factor proteins) changes according to the differentiation status of chondrocytes. This may be a likely scenario considering that that alternative splicing of COL2A1 is a developmentally regulated event during chondrogenesis. In fact, changes in expression of TIA-1 during Xenopus embryogenesis has been demonstrated (28), and, interestingly, expression of Xenopus type IIA procollagen was found to be similar to that of TIA-1 during the neurula stage of development (28, 29). We have also observed high levels of TIA-1 mRNA and protein in human embryonic kidney cells (results not shown), suggesting that the nuclear function of TIA-1 in regulating pre-mRNA splicing may be restricted to specific tissues in vivo in addition to specific phases of tissue development.

We determined that the TIA-1 binding site in the COL2A1 pre-mRNA AU-rich cis element consists of the U-rich sequence AUUUAUUUAUUU and that all three U-triplets are necessary for TIA-1 function in inducing exon 2 splicing (Fig. 2). This finding is consistent with previous reports of TIA-1 in regulating splicing of other genes by binding to U-rich regions downstream of alternative exons (9-12). In addition, it may be that the secondary structure of the COL2A1 cis element in addition to the primary sequence is also important for TIA-1 binding. TIA-1 has been reported to bind to RNA stem-loop conformations in virus RNA (30), whereas microarray and computational analyses have identified common U-rich TIA-1 binding motifs in human mRNA transcripts that are also predicted to form stem-loop structures (8). These studies analyzed only mature, spliced mRNA species, and it remains to be directly tested whether similar or different motifs are involved in TIA-1-dependent regulation of pre-mRNA splicing. With respect to the present study, more extensive and detailed analyses would be required to make any conclusions on secondary structure requirements for TIA-1 binding to the COL2A1 cis element described here.

TIA-1 also plays a functional role in the cytosol to down-regulate expression of inflammatory mediators by binding to AREs in the 3'-UTR of TNF- and COX-2 mRNA (22, 24). AREs, first discovered in 3'-UTRs of several cytokine and oncogene genes (31), are composed of one or more copies of the AUUUA pentamer or the UUAUUUAUU nonamer and play an important role in regulating message stability and translational efficacy (20). It is therefore interesting that an ARE-type sequence is present in the pre-mRNA intronic cis element described in the present study. The sequence of interest in COL2A1 pre-mRNA (AUUUAUUUAUUU) makes up half of the double-stranded stem region and is 100% conserved between species, and we have shown that TIA-1 is functional in binding to this sequence. We have also identified the same sequence in the 3'-UTR of COL2A1, and we will investigate if TIA-1 can also interact with this region. It may be that TIA-1 is also functional in controlling COL2A1 mRNA stability/translation by interacting with this ARE sequence in the 3'-UTR in addition to its role in regulating alternative splicing.

The present study has also shown that TIA-1 can bind to COL2A1 genomic DNA. To some of our RNA/TIA-1 binding experiments, we added competitor DNA probes as a control and, surprisingly, found that the DNA oligonucleotides completely inhibited binding of the AU-rich RNA oligonucleotide to TIA-1 (Fig. 4B). The sequence of the DNA probe corresponded to the wild-type AU-rich RNA cis element, and we subsequently showed that TIA-1 preferentially binds to the AT-rich single-stranded oligonucleotide rather than the double-stranded probe in vitro (Fig. 6A). Importantly, binding of endogenous TIA-1 to COL2A1 genomic DNA in vivo was confirmed by ChIP assays that included an additional RNase step (Fig. 7). This extra digestion step was necessary, because ChIP assays are also being used to identify endogenous RNA-binding proteins, because newly synthesized native RNA transcripts are also present in isolated chromatin complexes (32-34). In vitro DNA-binding activity has also been shown for the TIA-1-related protein, TIAR, by its ability to interact with a T-rich DNA probe corresponding to a region in the 3'-UTR of the VEGF gene (27). TIAR also shares similar properties with TIA-1 in binding to U-rich intronic regions to regulate pre-mRNA alternative splicing and in binding to AREs in 3'-UTRs of mRNA species thereby inhibiting their translation (24, 35-38). Similar to our findings with TIA-1, they also showed that TIAR preferentially binds to single-stranded rather than double-stranded DNA.

It is possible that the AT-rich cis element within intron 2 of COL2A1 genomic DNA is single-stranded in nature. This is not an uncommon phenomenon, because there have been numerous reports indicating regions of single-stranded DNA in or near regulatory regions of a variety of genes using chemical and enzymatic probes (39, 40). In addition, the RNA-binding protein hnRNP K1 has been shown to bind to a single-stranded region of genomic DNA within the c-myc gene promoter region to permit activation of transcription (41, 42). We have shown that the AU-rich RNA cis element contains secondary structure (19) (Fig. 1B), and it is possible that a similar intramolecular hairpin structure also exists in the AT-rich region in COL2A1 genomic DNA. Theoretical and NMR studies have predicted the existence of such DNA secondary structures (43, 44), whereas biochemical analyses have shown hairpin formation within an enhancer region of the human proenkephalin gene (45).

Our demonstration of endogenous binding of TIA-1 with a region of COL2A1 genomic DNA and the equivalent site in pre-mRNA suggests a novel, dual role for this nuclear protein in regulating processes of transcription and pre-mRNA alternative splicing by potentially shuttling between DNA and RNA ligands. There is a good deal of evidence in the published literature to show that processes of transcription and pre-mRNA splicing are tightly co-regulated in the nucleus (1, 46). For example, (i) transcription factors have been localized in spliceosome complexes (47, 48), (ii) splicing factors have been localized on the C-terminal domain of RNA polymerase II (49, 50), (iii) differences in promoter structure affects alternative splicing patterns (51, 52), (iv) introns are necessary for efficient RNA polymerase II-mediated transcription (53), (v) the transcriptional co-activator, p52, directly interacts with the ubiquitous splicing factor protein SF2/ASF (54), and (vi) splicing factor proteins can increase transcriptional elongation (55). Therefore, it is more than reasonable to suggest that a nuclear protein can carry out dual functions during transcription and splicing.

We postulate that during transcriptional activation of COL2A1, trans-acting factors (including TIA-1) are recruited to interact with specific cis regulatory motifs in genomic DNA. TIA-1 may bind to the AT-rich cis element in COL2A1 intron 2 described in the present study, and subsequently slow down RNA polymerase II and hence the rate of transcription. RNA polymerase II may eventually transcribe through this region, displacing TIA-1 from its DNA binding site, thus permitting TIA-1 to shuttle to the equivalent AU-rich ligand in the newly transcribed pre-mRNA region of COL2A1. TIA-1 can then carry out its enhancing function during alternative splicing to promote exon 2 inclusion as shown in the present study. This hypothesis is feasible, because it has been demonstrated that factors affecting the rate of RNA polymerase II-mediated transcriptional elongation can influence alternative splicing patterns, such that slower rates of transcription enhances inclusion of an alternative exon and vice versa (56). Our concept that RNA polymerase II may disrupt the TIA-1-DNA interaction was demonstrated in an in vitro transcription displacement assay (Fig. 8). This assay does not replicate the in vivo transcription process as the substrate DNA probe used was single-stranded and corresponds to the "coding" strand of genomic DNA that would not be transcribed in vivo. Instead, the aim of this experiment was to show that an RNA polymerase could disrupt TIA-1 binding to its single-stranded AT-rich DNA ligand. However, RNA polymerases (T7, Escherichia coli, and polymerase II) have been shown to interact with both the coding and template genomic DNA strands during transcription (57-59), which would favor our hypothesis of TIA-1 displacement by RNA polymerase II. In addition, single-stranded DNA conformations exist during active gene transcription where highly complex mechanisms control the process of chromatin remodeling that ultimately results in unwinding of duplex DNA by RNA polymerase II to provide a single-stranded template for synthesis of pre-mRNA (60). This may also explain our findings showing that TIA-1 interacts better with single-stranded rather than double-stranded DNA (Fig. 6).

In summary, we have demonstrated an additional role for TIA-1 in regulating alternative splicing via a unique AU-rich cis element and have predicted a new function for this splicing factor protein by its ability to interact with genomic DNA. It will be interesting to determine if TIA-1 can directly influence transcription of COL2A1 as well as other genes. This novel concept of a dual role in regulating transcription and pre-mRNA splicing may also be extended to other known nuclear RNA-binding proteins.

	FOOTNOTES

 
^* This work was supported by NIAMS, National Institutes of Health Grant AR036994 (to L. J. S. and A. M.) and an Arthritis Foundation Investigator grant (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Current Address: Dept. of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

¹ To whom correspondence should be addressed: Dept. of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Ave., Yalem 704, Box 8233, St Louis, MO 63110. Tel.: 314-454-8860; Fax: 314-454-5900; E-mail: mcalindena{at}wustl.edu.

³ The abbreviations used are: pre-mRNA, precursor messenger RNA; TIA-1, T-cell-restricted intracellular antigen-1; COL2A1, type II procollagen gene; ChIP, chromatin immunoprecipitation; RRM, RNA recognition motif; ARE, AU-rich element; UTR, untranslated region; TIAR, TIA-1-related protein; GST, glutathione S-transferase; IP, immunoprecipitation; RT, reverse transcription.

⁴ M. Goldring, personal communication.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Kornblihtt, A. R., de la Mata, M., Fededa, J. P., Munoz, M. J., and Nogues, G. (2004) RNA (N. Y.) 10, 1489-1498[CrossRef]
2. Derrigo, M., Cestelli, A., Savettieri, G., and Di Liegro, I. (2000) Int. J. Mol. Med. 5, 111-123[Medline] [Order article via Infotrieve]
3. Dreyfuss, G., Kim, V. N., and Kataoka, N. (2002) Nat. Rev. Mol. Cell. Biol. 3, 195-205[CrossRef][Medline] [Order article via Infotrieve]
4. Kawakami, A., Tian, Q., Streuli, M., Poe, M., Edelhoff, S., Disteche, C. M., and Anderson, P. (1994) J. Immunol. 152, 4937-4945[Abstract]
5. Beck, A. R., Medley, Q. G., O'Brien, S., Anderson, P., and Streuli, M. (1996) Nucleic Acids Res. 24, 3829-3835[Abstract/Free Full Text]
6. Dember, L. M., Kim, N. D., Liu, K. Q., and Anderson, P. (1996) J. Biol. Chem. 271, 2783-2788[Abstract/Free Full Text]
7. Zhang, T., Delestienne, N., Huez, G., Kruys, V., and Gueydan, C. (2005) J. Cell Sci. 118, 5453-5463[Abstract/Free Full Text]
8. Lopez de Silanes, I., Galban, S., Martindale, J. L., Yang, X., Mazan-Mamczarz, K., Indig, F. E., Falco, G., Zhan, M., and Gorospe, M. (2005) Mol. Cell. Biol. 25, 9520-9531[Abstract/Free Full Text]
9. Del Gatto-Konczak, F., Bourgeois, C. F., Le Guiner, C., Kister, L., Gesnel, M. C., Stevenin, J., and Breathnach, R. (2000) Mol. Cell. Biol. 20, 6287-6299[Abstract/Free Full Text]
10. Forch, P., Puig, O., Kedersha, N., Martinez, C., Granneman, S., Seraphin, B., Anderson, P., and Valcarcel, J. (2000) Mol. Cell 6, 1089-1098[CrossRef][Medline] [Order article via Infotrieve]
11. Le Guiner, C., Lejeune, F., Galiana, D., Kister, L., Breathnach, R., Stevenin, J., and Del Gatto-Konczak, F. (2001) J. Biol. Chem. 276, 40638-40646[Abstract/Free Full Text]
12. Zuccato, E., Buratti, E., Stuani, C., Baralle, F. E., and Pagani, F. (2004) J. Biol. Chem. 279, 16980-16988[Abstract/Free Full Text]
13. Burge, C. B., Tuschl, T., and Sharp, P. A. (1999) in The RNA World, 2nd Ed., (Gesteland, R., Cech, T., and Atkins, J., eds) pp. 525-560, Cold Spring Harbor Press, Cold Spring Harbor, New York
14. Forch, P., Puig, O, Martinez, C., Seraphin, B., and Valcarcel, J. (2002) EMBO J. 21, 6882-6892[CrossRef][Medline] [Order article via Infotrieve]
15. Ala-Kokko, L., Kvist, A. P., Metsaranta, M., Kivirikko, K. I., de Crombrugghe, B., Prockop, D. J., and Vuorio, E. (1995) Biochem. J. 308, 923-929[Medline] [Order article via Infotrieve]
16. Ng, L. J., Tam, P. P., and Cheah, K. S. (1993) Dev. Biol. 159, 403-417[CrossRef][Medline] [Order article via Infotrieve]
17. Sandell, L. J., Nalin, A. M., and Reife, R. A. (1994) Dev. Dyn. 199, 129-140[Medline] [Order article via Infotrieve]
18. Ryan, M. C., and Sandell, L. J. (1990) J. Biol. Chem. 265, 10334-10339[Abstract/Free Full Text]
19. McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711[Abstract/Free Full Text]
20. Zhang, T., Kruys, V., Huez, G., and Gueydan, C. (2002) Biochem. Soc. Trans 30, 952-958[CrossRef][Medline] [Order article via Infotrieve]
21. Barreau, C., Paillard, L., and Osborne, H. B. (2005) Nucleic Acids Res. 33, 7138-7150[CrossRef][Medline] [Order article via Infotrieve]
22. Piecyk, M., Wax, S., Beck, A. R., Kedersha, N., Gupta, M., Maritim, B., Chen, S., Gueydan, C., Kruys, V., Streuli, M., and Anderson, P. (2000) EMBO J. 19, 4154-4163[CrossRef][Medline] [Order article via Infotrieve]
23. Phillips, K., Kedersha, N., Shen, L., Blackshear, P. J., and Anderson, P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2011-2016[Abstract/Free Full Text]
24. Cok, S. J., Acton, S. J., Sexton, A. E., and Morrison, A. R. (2004) J. Biol. Chem. 279, 8196-8205[Abstract/Free Full Text]
25. Kawai, T., Lal, A., Yang, X., Galban, S., Mazan-Mamczarz, K., and Gorospe, M. (2006) Mol. Cell. Biol. 26, 3295-3307[Abstract/Free Full Text]
26. Tenenbaum, S. A., Lager, P. J., Carson, C. C., and Keene, J. D. (2002) Methods 26, 191-198[CrossRef][Medline] [Order article via Infotrieve]
27. Suswam, E. A., Li, Y. Y., Mahtani, H., and King, P. H. (2005) Nucleic Acids Res. 33, 4507-4518[Abstract/Free Full Text]
28. Rothe, F., Gueydan, C., Bellefroid, E., Huez, G., and Kruys, V. (2006) Biochem. Biophys. Res. Commun. 343, 57-68[CrossRef][Medline] [Order article via Infotrieve]
29. Larrain, J., Bachiller, D., Lu, B., Agius, E., Piccolo, S., and De Robertis, E. M. (2000) Development 127, 821-830[Abstract]
30. Li, W., Li, Y., Kedersha, N., Anderson, P., Emara, M., Swiderek, K. M., Moreno, G. T., and Brinton, M. A. (2002) J. Virol. 76, 11989-12000[Abstract/Free Full Text]
31. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1670-1674[Abstract/Free Full Text]
32. Abruzzi, K. C., Lacadie, S., and Rosbash, M. (2004) EMBO J. 23, 2620-2631[CrossRef][Medline] [Order article via Infotrieve]
33. Lacadie, S. A., and Rosbash, M. (2005) Mol. Cell 19, 65-75[CrossRef][Medline] [Order article via Infotrieve]
34. Listerman, I., Sapra, A. K., and Neugebauer, K. M. (2006) Nat. Struct. Mol. Biol. 13, 815-822[CrossRef][Medline] [Order article via Infotrieve]
35. Kawakami, A., Tian, Q., Duan, X., Streuli, M., Schlossman, S. F., and Anderson, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8681-8685[Abstract/Free Full Text]
36. Zhu, H., Hasman, R. A., Young, K. M., Kedersha, N. L., and Lou, H. (2003) Mol. Cell. Biol. 23, 5959-5971[Abstract/Free Full Text]
37. Mazan-Mamczarz, K., Lal, A., Martindale, J. L., Kawai, T., and Gorospe, M. (2006) Mol. Cell. Biol. 26, 2716-2727[Abstract/Free Full Text]
38. Gueydan, C., Droogmans, L., Chalon, P., Huez, G., Caput, D., and Kruys, V. (1999) J. Biol. Chem. 274, 2322-2326[Abstract/Free Full Text]
39. Grosso, L. E., and Pitot, H. C. (1985) Cancer Res. 45, 5035-5041[Abstract/Free Full Text]
40. Ma, D., Xing, Z., Liu, B., Pedigo, N. G., Zimmer, S. G., Bai, Z., Postel, E. H., and Kaetzel, D. M. (2002) J. Biol. Chem. 277, 1560-1567[Abstract/Free Full Text]
41. Tomonaga, T., and Levens, D. (1995) J. Biol. Chem. 270, 4875-4881[Abstract/Free Full Text]
42. Tomonaga, T., and Levens, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5830-5835[Abstract/Free Full Text]
43. Ikuta, S., Chattopadhyaya, R., Ito, H., Dickerson, R. E., and Kearns, D. R. (1986) Biochemistry 25, 4840-4849[CrossRef][Medline] [Order article via Infotrieve]
44. Erie, D. A., Suri, A. K., Breslauer, K. J., Jones, R. A., and Olson, W. K. (1993) Biochemistry 32, 436-454[CrossRef][Medline] [Order article via Infotrieve]
45. McMurray, C. T., Juranic, N., Chandrasekaran, S., Macura, S., Li, Y., Jones, R. L., and Wilson, W. D. (1994) Biochemistry 33, 11960-11970[CrossRef][Medline] [Order article via Infotrieve]
46. Cramer, P., Srebrow, A., Kadener, S., Werbajh, S., de la Mata, M., Melen, G., Nogues, G., and Kornblihtt, A. R. (2001) FEBS Lett. 498, 179-182[CrossRef][Medline] [Order article via Infotrieve]
47. Zhou, Z., Licklider, L. J., Gygi, S. P., and Reed, R. (2002) Nature 419, 182-185[CrossRef][Medline] [Order article via Infotrieve]
48. Davies, R. C., Calvio, C., Bratt, E., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217-3225[Abstract/Free Full Text]
49. Yuryev, A., Patturajan, M., Litingtung, Y., Joshi, R. V., Gentile, C., Gebara, M., and Corden, J. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6975-6980[Abstract/Free Full Text]
50. Du, L., and Warren, S. L. (1997) J. Cell Biol. 136, 5-18[Abstract/Free Full Text]
51. Cramer, P., Pesce, C. G., Baralle, F. E., and Kornblihtt, A. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11456-11460[Abstract/Free Full Text]
52. Cramer, P., Caceres, J. F., Cazalla, D., Kadener, S., Muro, A. F., Baralle, F. E., and Kornblihtt, A. R. (1999) Mol. Cell 4, 251-258[CrossRef][Medline] [Order article via Infotrieve]
53. Furger, A., O'Sullivan, J. M., Binnie, A., Lee, B. A., and Proudfoot, N. J. (2002) Genes Dev. 16, 2792-2799[Abstract/Free Full Text]
54. Ge, H., Si, Y., and Wolffe, A. P. (1998) Mol. Cell 2, 751-759[CrossRef][Medline] [Order article via Infotrieve]
55. Fong, Y. W., and Zhou, Q. (2001) Nature 414, 929-933[CrossRef][Medline] [Order article via Infotrieve]
56. Kornblihtt, A. R. (2006) Nat. Struct. Mol. Biol. 13, 5-7[CrossRef][Medline] [Order article via Infotrieve]
57. Shi, Y. B., Gamper, H., and Hearst, J. E. (1988) J. Biol. Chem. 263, 527-534[Abstract/Free Full Text]
58. Shi, Y. B., Gamper, H., Van Houten, B., and Hearst, J. E. (1988) J. Mol. Biol. 199, 277-293[CrossRef][Medline] [Order article via Infotrieve]
59. Kettenberger, H., Armache, K. J., and Cramer, P. (2004) Mol. Cell 16, 955-965[CrossRef][Medline] [Order article via Infotrieve]
60. Saunders, A., Core, L. J., and Lis, J. T. (2006) Nat. Rev. Mol. Cell. Biol. 7, 557-567[CrossRef][Medline] [Order article via Infotrieve]

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