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J. Biol. Chem., Vol. 279, Issue 14, 13668-13676, April 2, 2004

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TIA Proteins Are Necessary but Not Sufficient for the Tissue-specific Splicing of the Myosin Phosphatase Targeting Subunit 1^*

Supriya Shukla, Wessel P. Dirksen, Katherine M. Joyce, Caroline Le Guiner-Blanvillain, Richard Breathnach, and Steven A. Fisher¶||

From the Departments Of Medicine (Cardiology) and ^¶Physiology and Biophysics, Case Western Reserve University school of Medicine, Cleveland, Ohio 44106-4958 and INSERM U463, Institute de Biologie-CHR, 9 Quai Moncousu, 44093 Nantes Cedex 1, France

Received for publication, December 24, 2003 , and in revised form, January 16, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1^-/-TIAR^+/-) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC₁₇ alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Smooth muscle (SM)¹ tissues show considerable phenotypic and functional diversity. Much phenotypic diversity in SM tissues is generated by the alternative splicing of exons (1–4). Nearly 60% of vertebrate genes are estimated to undergo alternative splicing (5, 6). Whereas there are a few well characterized genes where alternative splicing is governed by the presence or absence of a single regulatory protein (6), in many genes regulated splicing of exons involves a combinatorial interplay of positive and negative cis-regulatory elements and trans-acting proteins (3, 7). These regulatory interactions affect the splice site selection by the splicing machinery and thus govern the inclusion or exclusion of alternative exons.

Our laboratory is using the MYPT1 gene (a subunit of smooth muscle myosin phosphatase) as a model gene to understand how SM phenotypic diversity is generated through alternative splicing of exons for several reasons. In birds and mammals, isoforms of MYPT1 are generated by cassette-type alternative splicing of exons in the central and 3'-end of the transcript. The splicing of the alternative exons is tissue-specific and developmentally regulated. Around the time of hatching (embryonic day 21 (ED21)), the MYPT1 isoform in the chicken gizzard (phasic fast contracting smooth muscle phenotype) completely switches from central exon-in and 3'-exon-out to central exon-out and 3'-exon-in. In contrast, the chicken slow tonic contracting aorta smooth muscle phenotype throughout development expresses the MYPT1 isoform in which the central alternative exon is included and the 3' alternative exon is skipped (8, 9). The tissue-specific expression of the MYPT1 splice variants is thought to be an important determinant of smooth muscle functional diversity (9, 10).

We previously used mutation and deletion analysis of MYPT1 minigene constructs to identify cis-regulators of splicing of the chicken MYPT1 central alternative exon (from here on referred to as the MYPT1 alt exon). We identified several cis-enhancer elements (Fig. 1B) in the vicinity of the alternative exon 5'-splice site, including a proximal U-rich intronic sequence, a 67-nucleotide intronic enhancer, and an exonic splicing enhancer, that are necessary for the splicing of the MYPT1 alt exon (11). The proximal intronic splicing enhancer (IE1) is a 19-nucleotide U-rich sequence including two UCUU motifs immediately adjacent to the 5' splice site of the alternative exon. This sequence is a putative binding site for TIA-1 and TIAR (12–14) and PTB (15) proteins, suggesting a possible role of these proteins in the regulation of MYPT1 alt exon splicing. TIA-1 and TIAR are RNA-binding proteins regulating a number of functions in both nucleus and cytoplasm (16, 17). They have been shown to be the activators of constitutive as well as alternative splicing (12, 14, 18–20). PTB, also known as heterogeneous nuclear ribonucleoprotein-I, is a well known repressor of splicing (21). In the current study, we use gain and loss of function experiments as well as RNA-protein binding experiments to show that TIA-1 or TIAR functioning through the U-rich enhancer element is necessary but not sufficient for the splicing of the MYPT1 alt exon. We also find that expression of the TIA proteins is developmentally regulated, suggesting a model in which the silencing of TIA expression during the development of the phasic fast (gizzard) smooth muscle phenotype contributes to the loss of splicing of the MYPT1 alt exon.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Diagram of MYPT1 minigene expression construct and mutations and deletions. A, the MYPT1 minigene is shown. Solid box, alternative exon; open boxes, flanking exons. The horizontal lines connecting these boxes represent introns. Sizes of exons and introns are not drawn to scale. Upstream is the Rous sarcoma virus promoter, and downstream is the bovine growth hormone polyadenylation signal (gray box) in the pRcRSV plasmid. The arrows indicate the approximate locations of the oligonucleotides that were used in all of the RT-PCRs in this study. B, diagram of previously identified cis-regulators of splicing of the alternative exon. WT sequences are shown on the first line with regulatory sequences in boldface type. Deletions and mutations of the IE1 (U-rich sequence) and ESE are shown below the WT sequence. The ESE contains an 11-nt perfect palindromic sequence (boldface type), which is mutated to destroy the putative SR protein-binding site (Mut pal1) or to destroy both half-sites of the palindromic sequence (Mut pal1 + 2). Mut A alters the sequence immediately upstream of the palindrome. These mutation and deletion constructs were previously created and characterized (11).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture, Electroporation, and RNA Isolation from DT-40 Cells— DT40 cells were cultured and electroporated as described by Le Guiner et al. (20). For electroporations, 30 µg of a minigene construct were mixed with 3 µg of a plasmid carrying a blasticidin resistance marker. Cells were selected in medium containing 50 µg/ml blasticidin, and RNA was prepared and reverse-transcribed as described by Le Guiner et al. (20).

RT-PCR of RNA—RNA samples were analyzed by RT-PCR for the presence of exon-included and exon-skipped MYPT-1 minigene transcripts as described previously (11). In these reactions, a 3' primer is used that specifically anneals with the bovine growth hormone polyadenylation sequence present only in the exogenous MYPT1 minigene transcript. The RT-PCR products were separated on a 6% native polyacrylamide gel, and fluorescent signals of the exon-included and exon-excluded products were quantified with a Storm 860 Imager (Amersham Biosciences) and Amersham Biosciences ImageQuaNT software. Splice variants of MLC₁₇, MYPT1 central, and MYPT1 C-terminal were examined in DT40 cells and DT40 TIA^-/-TIAR^+/- (referred as TIAmut cells hereon) (20) using primers as previously described (11). For RT-PCR, 23–27 (for transfected gizzard cells), 30–35 (for endogenous genes from DT40 cells), and 60–65 (for DT40 stably transfected cells) cycles of amplification were used. RT reactions were done with 0.5–1.5 µg of total RNA, and one-twentieth to one-one hundredth of the RT reaction used in the PCR. For every sample, negative control reactions were performed in which the avian myeloblastosis virus RT enzyme was omitted from the RT reaction. To demonstrate the linearity of the assays, PCRs were performed using 0.5 and 1.5 µl of input cDNA from each RT reaction. As previously shown (11), this resulted in severalfold increase in the total signal without any significant change in the ratio of the exon-included to exon-skipped products (not shown).

Preparation of Nuclear and Whole Tissue Extracts—Nuclear and whole tissue extracts were prepared from embryonic and posthatched chick gizzard and aorta tissues using the Nuclear Extract Kit (Active Motif, Carlsbad, CA) as described by the manufacturer. Samples were assayed for protein concentrations using BCA protein assay kit (Pierce).

Synthesis of Labeled RNA Oligonucleotides—The 5'-end amine-modified RNA oligonucleotides containing a wild type or a mutant IE1 or an ESE sequence were purchased from Oligos Etc. (Wilsonville, OR). The oligonucleotides were biotin-labeled with sulfo-succin-midyl-6'-biotinamido-hexanoate by Pierce, following the procedure as described by the manufacturer. The labeled oligonucleotides were purified by ethanol precipitation followed by polyacrylamide gel purification on a 19% gel.

RNA Affinity Assays—RNA affinity assay was carried out following the method described by Blyn et al. (22). Briefly, binding reactions were performed in 250 µl of binding buffer consisting of 100 mM HEPES, 10 mM MgCl₂, 500 mM KCl, 50% glycerol, 10 mM dithiothreitol, 20 mM creatine phosphate, 2 mM ATP, and 2.0 µl of bovine serum albumin (10 mg/ml), 10 µg t-RNA, 200 pmol of biotin-labeled RNA, and 250 or 400 µg of NE. After a 10-min incubation at 30 °C, the reaction mixture was added to 400 µl of streptavidin Magnesphere particles (Promega, Madison, WI), prewashed five times with the binding buffer without t-RNA. The reaction mixture was incubated with prewashed beads for 15 min at 30 °C to allow binding of the protein-RNA complex to the beads. The beads-RNA-protein complex was then washed five times with binding buffer. After final wash, 30 µl of 2x protein sample buffer was added to the complex and incubated for 10 min at 30 °C to allow dissociation of the protein from the complex. 10 µl of each protein sample (one-third of the total sample) was analyzed by SDS-PAGE and Western blotting.

Protein Analysis—Proteins were separated on 4–12% gradient Nu-PAGE Novex bis-tris gels (Invitrogen) and either visualized by silver stain or electroblotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). Membranes were blocked in 5% nonfat dry milk (w/v) in wash buffer for 1 h and probed with antibodies ML 29 against TIA-1, 6E3 against TIAR (23), and mAb 104 against SR proteins (24) and SF2-specific antibody (25). The secondary antibody was a goat anti-mouse IgG conjugated with horseradish peroxidase (1:1600; Bio-Rad). Signals were detected using the ECL chemiluminescence kit (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
TIA-1 and TIAR trans-Activate MYPT1 Central Exon Splicing—Transfection of the MYPT1 wild type minigene expression plasmid (Fig. 1A) into cultured gizzard SMCs resulted in 89 ± 2% exon-included transcripts as we previously reported (11) (Fig. 2A). Co-transfection of a TIA-1 expression plasmid increased the level of exon-included product from 89 ± 2% to 98 ± 2% (Fig. 2A, lanes 1 and 2). Co-transfection of a TIAR expression plasmid had a similar but slightly less potent effect (95 ± 3% in wild type Fig. 2A, lane 3). In contrast, co-transfection with a PTB expression plasmid resulted in a small shift toward the exon-exclusion in the cultured SMCs (85 ± 4%, Fig. 2A, lane 4).

View larger version (32K): [in this window] [in a new window]   FIG. 2. TIA proteins promote inclusion of MYPT1 alt exon. Gizzard SMCs (A) and 293 cells (nonmuscle cells) (B) were co-transfected in 24-well plates with the wild type MYPT1 minigene plasmid (0.1 µg) with or without an expression plasmid for TIA-1/TIAR or PTB (three different doses of each plasmid, 0.01, 0.05, or 0.1 µg). RNA was harvested 24 h later, and ratios of MYPT1 exon-in to exon-out minigene transcript ratios were determined by RT-PCR followed by PAGE. In this and subsequent figures, a picture of a representative gel is shown below, which is the quantification from at least three independent transfections. Data are shown as the mean ± S.D. Labels on the left refer to the exon-included (407-bp) and exon-skipped (284-bp) RT-PCR products. n.s., nonspecific RT-PCR products. -RT, control reactions performed with all samples in which RT enzyme was omitted. At the top is a schematic diagram of the MYPT1 minigene construct used for transfections.

trans-Activation Effect of TIA Is U-rich IE1-dependent—To test whether the observed enhancer effect of TIA on splicing is functioning through the U-rich IE1, TIA-1 or TIAR expression plasmids were co-transfected with MYPT1 minigene constructs where the IE1 is deleted (UCUU1 + 2) or mutated (Cmut2 and Cmut1 + 2) (Fig. 1B). Deletion of the 19-nt U-rich sequence results in a significant loss of exon splicing in cultured SMCs (19 ± 4% exon inclusion) (see Fig. 3A, lane 3). Co-transfection with the TIA-1 expression plasmid had no effect on exon splicing (15 ± 2% exon-in) (Fig. 3A, lanes 3 and 4). Similarly, mutation of the U-rich sequence to C-rich (Cmut1 + 2) also resulted in exon skipping and failure of TIA-1 co-transfection to enhance splicing (15 ± 2% versus 18 ± 4%) (Fig. 3A, lanes 5 and 6). When the U-rich sequence was partially mutated (Cmut2), a smaller shift toward exon exclusion occurred, and this could be entirely rescued by co-expression of TIA-1 (60 ± 4 versus 92 ± 2% exon-included) (Fig. 3A, compare lanes 7 and 8). Nearly identical results were obtained with co-transfection of a TIAR expression plasmid and the MYPT1 mini-gene constructs (data not shown). Thus, TIA-1 and TIAR trans-activation of MYPT1 alt exon splicing is IE1-dependent.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Activation of MYPT1 alt exon splicing by TIA is IE1- and ESE-dependent but independent of IE2. A, a wild type MYPT1 minigene or mutants where IE1 is either deleted or mutated (0.01 µg) was co-transfected with an expression plasmid for TIA-1 in gizzard cells. B, a mutant MYPT1 minigene where ESE or IE2 is either deleted or mutated (0.01 µg) was co-transfected with an expression plasmid for TIA-1 (0.01, 0.05, or 0.1 µg) in gizzard cells. Deletions and mutations are shown in Fig. 1. Chimera refers to complete replacement of alt exon with corresponding rat (constitutively spliced) exon (11). Methods were as described in the legend to Fig. 2. In this and subsequent experiments, similar results were obtained with co-transfection of TIAR expression plasmids (not shown).

To more specifically define the co-activator, we tested the effects of mutation of two previously identified cis-enhancers. A mutation that disrupts a palindromic ESE (Mut pal1 + 2) (Fig. 1) resulted in a reduction in exon inclusion to 49 ± 3% as previously reported. This mutation disrupts both half-sites of the palindromic ESE as well as the putative SRp55 binding sequence (UACAUC). Co-transfection of a TIA expression plasmid had no significant effect on alt exon splicing (45 ± 4%) (see Fig. 3B, compare lanes 3 and 4). A more limited mutation of the 5'-half of the palindromic ESE (Mut pal1, Fig. 1), which destroys the putative binding site for SRp55, has a more modest effect on exon splicing but also does not respond to co-transfection with TIA vector (Mut pal 1 75 ± 2% versus 76 ± 3% exon inclusion) (Fig. 3B, lanes 5 and 6). In contrast, mutation of 10 nt immediately upstream of the palindromic ESE to the corresponding rat sequence that resulted in a significant loss in exon inclusion could be completely rescued by co-transfection with a TIA expression plasmid (mutA; 59 ± 3% versus 88 ± 2% exon inclusion) (Fig. 3B, lanes 7 and 8). In contrast to the requirement of the ESE for TIA function in alt exon splicing, the effect of deletion of 661 nt in the downstream intron that contains a 67-nt enhancer (IE2) was also completely rescued by co-transfection of a TIA expression plasmid (1, 65 ± 5% versus 93 ± 2% exon-in) (Fig. 3B, compare lanes 9 and 10). Thus, TIA function to enhance splicing of the alt exon is dependent upon the palindromic ESE but not an adjacent ESE nor downstream IE2.

Effect of TIA-1 and TIAR Gene Disruption on MYPT1 Alt Exon Splicing—We next examined the effect that TIA loss-of-function would have on MYPT1 alt exon splicing. MYPT1 is expressed in nonmuscle and SM cells, and the central alt exon is spliced in nonmuscle cells. This allowed us to utilize the chicken lymphoid DT40 cell line in which both TIA genes were intact (WT) or with both TIA-1 alleles and one TIAR allele disrupted (TIA-1^-/-TIAR^+/-) cells (TIAmut) (20). Stable transfection of wild type DT40 cells with the wild type MYPT1 minigene generated 88 ± 2% exon-included product (Fig. 4, lane 1), identical to that obtained with transient transfection of cultured chicken SMCs and the chicken hepatocellular carcinoma LMH cell line (11). The MYPT1 minigene construct stably transfected into DT40 TIAmut cells resulted in a large shift toward exon skipping (54 ± 5% exon inclusion) (Fig. 4, lane 2). This effect is similar to the effect of partially disabling the TIA cis-element Cmut2 (see Fig. 3A, lane 7;60 ± 4% exon inclusion) and suggests that the remaining TIAR allele can partially support MYPT1 alt exon splicing, consistent with the co-transfection assays. Stable transfection of DT40 WT with the Cmut1 + 2 MYPT1 minigene plasmid resulted in a large shift toward exon skipping with only 15 ± 3% exon-included transcripts (Fig. 4, lane 3), similar to that observed in the transient transfection of smooth and nonmuscle cells. This same construct in the TIAmut DT40 cells generated 5 ± 4% exon-included transcripts (Fig. 4, lane 4), suggesting that the U-rich flanking sequence remaining in this construct has residual TIA-dependent enhancer activity (consistent with TIA protein-binding studies; see below).

View larger version (31K): [in this window] [in a new window]   FIG. 4. Genetic depletion of TIA proteins diminishes splicing of the MYPT1 alt exon. The chicken lymphoid DT40 cell line, wild type, and TIA-/-TIAR+/- cells (TIAmut), were stably transfected with wild type or mutant MYPT1 minigene plasmids as described under "Materials and Methods." RNA was harvested, and ratios of MYPT1 exon-in to exon-out minigene transcript ratios determined as described in the legend to Fig. 2.

View larger version (27K): [in this window] [in a new window]   FIG. 5. TIA proteins bind to IE1, and SRp55 and SRp20 bind to the ESE. An RNA affinity binding assay was carried out with biotinylated RNA oligonucleotides containing a 19-nt wild-type or mutant IE1 sequence, the ESE sequence, or beads only (A). NEs for binding reactions were collected from four different stages of chicken gizzard development (ED15, ED19, D1, and D6). Biotinylated RNA was incubated with the NE under binding conditions (see "Materials and Methods"). Nuclear proteins bound to the RNA were mixed with streptavidin columns, and bound proteins were eluted and analyzed by SDS-PAGE and Western blotting as described under "Materials and Methods." The oligonucleotide containing the IE1 served as a control for the ESE binding reaction (C) and vice versa. Nuclear extracts were also mixed with SA beads (beads alone) as a control for nonspecific binding. B, a silver stain and Western blot analysis of proteins binding to the IE1 sequence using ED15 NE. Western blot analysis was carried out using anti-TIA-1 antibody ML29 and anti-TIAR 6E3 antibody (not shown). C, Western blot analysis of TIA-IE1 binding using NEs from different developmental stages, as described above. A silver stain of total NEs used in this study is also shown. D, a Western blot analysis of purified proteins binding to the ESE, using SR protein-specific mAb 104. Nuclear extracts were used from different developmental stages (described above).

TIA Binding to IE1 and SR Binding to ESE Follows the Developmentally Regulated Pattern of MYPT1 Alt Exon Splicing—We next sought to determine whether there was a relationship between binding of the TIA and SR proteins to the enhancer sequences and the developmental switch from MYPT1 alt exon splicing to exon exclusion in the gizzard smooth muscle. Affinity binding assays were carried out using the NEs collected from four different periods of development with the following splicing pattern of MYPT1 alt exon: 1) ED15–100% exon-included; 2) ED19–78% exon included; 3) D1–40% exon-included; 4) D6–10% exon-included. No significant difference was observed in the binding of TIA to the IE1 between the NE from ED15 and ED19 (Fig. 5, B (lane 5) and C (lane 1)). However, there was a dramatic decrease in TIA-IE1 binding in NE from D1 (Fig. 5C, lane 3). No binding of TIA proteins was detected with the NE from D6 tissue (Fig. 5C, lane 5), even when 4-fold increased amounts of nuclear proteins were used in the binding reaction. A silver stain of the NEs used in these experiments (Fig. 5C, lanes 7–9) shows approximately equal amounts of intact protein in each sample. A similar pattern was observed for SRp55 and SRp20 binding to the ESE (Fig. 5D, compare lanes 1, 3, 5, and 7).

Down-regulated Expression of TIA and SR Proteins in Developing Chick Gizzard Correlates with Developmental Changes in MYPT1 Central Exon Splicing—The stage-dependent binding of TIA and SR proteins to the IE1 and ESE, respectively, prompted us to examine the expression of the TIA and SR proteins during smooth muscle phenotypic specification. We determined the abundance of these proteins in whole tissue extracts (WTEs) and nuclear fractions from ED15, ED19, D1, and D6 chicken gizzard. In NEs from ED15 and ED19, TIA-1 was detected as a singlet band and TIAR as a doublet at 42 kDa (Fig. 6A, lanes 1 and 2), as previously reported in avian species (20). Additionally, in ED19, D1, and D6 NEs, a band migrating at 55 kDa was also observed. This band of unknown identity has been previously reported (28, 29). There was a significant decline in the levels of expression of TIA1 and TIAR proteins in the gizzard from days 1–6 after hatching, when examined in WTEs or nuclear subfraction protein extracts. Silver staining of protein extract showed similar amounts of intact protein in each sample (Fig. 5C). When 4-fold more of the D6 nuclear extract protein (40 µg) was loaded onto a gel, weak signals for TIA1 and TIAR were observed (not shown). We also examined the expression of SR proteins in whole tissue extracts, nuclear extracts, and purified SR preparations (24) from gizzard tissues spanning the same developmental period. Western blot analysis of WTEs using the antibody mAb 104 for SR proteins revealed abundance of SRp55, SRp40, and SRp30 in the samples. SRp30 proteins appeared as a doublet. The slower migrating band (SRp30A) was identified as SF2 with a specific monoclonal antibody (25) (not shown). SRp20 and SRp75 were much less abundant but also were present at all stages of development. Similar patterns were observed in nuclear extracts and purified SR preparations probed with either the mAb 104 or SF2-specific antibodies (not shown). In either the whole tissue or subfraction extracts, there was a 2–4-fold decrease in the abundance of the SR proteins from ED19 to the adult gizzard. There were no significant changes in the relative abundance of each SR protein within a sample.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Down-regulated expression of TIA and SR proteins during chicken gizzard development. Western blot analysis of TIA-1, TIAR, and SR proteins in WTEs (20 µg/sample) and NEs (10 µg/sample) from ED11–ED15 to adult gizzard. A, Western blot analysis of WTEs isolated from ED15, ED19, D1, and D6 gizzard tissues using anti-TIA-1 ML29 (top), anti-TIAR 6E3 (middle), and anti-GAPDH (bottom; as an internal control) antibodies. B, Western blot analysis of gizzard tissue NEs prepared from the stages described above, using anti-TIA ML29 (top) or anti-TIAR 6E3 (bottom). Silver-stained gel of these protein samples is shown in Fig. 5. C, Western blot analysis of WTEs of gizzard isolated from ED11, ED15, ED21, and adult chicken using SR protein-specific mAb 104 antibody (top) and -actin antibody (bottom; as an internal control). Similar results were obtained with nuclear extracts or purified SR protein preparations from these tissues (not shown).

View larger version (38K): [in this window] [in a new window]   FIG. 7. Genetic depletion of TIA proteins specifically diminishes splicing of endogenous MYPT1 alt exon. RNA was isolated from DT40 wild type cells and DT40 TIAmut cells (TIA1-/-TIAR+/-) and analyzed for exon-in exon-out ratios of endogenous MYPT-1 central alternative exon (A), MYPT-1 3' alternative exon (B), or MLC17 alternative exon (C). Oligonucleotides used in these reactions were previously described. The percentage of exon inclusion observed is shown below the lanes. The control reaction represents the same RT-PCR with chicken smooth muscle samples that are known to give exon-in and/or exon-out products. The exon-included and exon-excluded products in A, B, and C are 450 and 325 bp, 461 and 422 bp, and 347 and 316 bp, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TIA-1/TIAR Binds to U-rich IE1 and Activates MYPT1 Alt Exon Splicing in an ESE-dependent Manner—We previously identified a 19-nt U-rich sequence just downstream of the alternative exon 5' splice site that is necessary for splicing of the alternative exon. This sequence also contains two UCUU sequences that are consensus for PTB binding (15). In this study, we show that co-transfection of TIA-1 or TIAR expression vectors increases exon inclusion, whereas co-transfection of PTB has minimal effect, suggesting that TIA-1 and TIAR function in a redundant manner to enhance MYPT1 alt exon splicing. That TIA-1 and TIAR are redundant in this context is also suggested by the loss-of-function experiments. Co-transfection of a dominant negative TIA-1 plasmid construct with the WT MYPT1 construct into 293 cells had no effect on exon splicing despite nearly complete elimination of TIA-1 expression, presumably due to continued expression of TIAR. In DT40 cells, complete abrogation of TIA-1 expression and partial abrogation of TIAR resulted in a 35% reduction in splicing of the endogenous alt exon. Thus, splicing of the endogenous alt exon is TIA-dependent, and the presence of even one of four functional TIA alleles can support splicing of the alt exon. The effect of inactivation of all four TIA alleles could not be studied in this system, since it results in cell lethality and will thus require a conditional inactivation approach. Interestingly, a partial mutation of the U-rich cis-element sequence in the minigene construct produces the same approximate effect on alt exon splicing (35% exon out) as occurs with the inactivation of three of four TIA alleles (35% exon out), and there was generally a close relationship between the effects of mutations on TIA binding assays and exon splicing.

TIA-1 and TIAR genes have also been inactivated in mice. Mice with both TIA-1- and TIAR-disrupted genes die before embryonic day 7, whereas mice with disrupted TIAR show 90% embryonic lethality (17). TIA-1 and TIAR inactivation produces different developmental phenotypes in mice. This may be due to the unique patterns of expression in developing mouse tissues or alternatively to nonredundant functions that were not evident in our in vitro experiments. TIA proteins also are involved in translational control, stress responses, and regulation of cell death, and it remains to be determined which of these functions are required for cell and embryo viability (17, 23, 31). Further, TIAR-deficient mice fail to develop spermatogonia, indicating that TIAR is important for primordial germ cell activity (17, 29).

In this and a prior study, we have shown that TIA is necessary but not sufficient for splicing of the MYPT1 central alternative exon. Mutation of an 11-nt perfect palindromic sequence just upstream of the 5' splice site and/or additional 5' sequence significantly reduces splicing of the alt exon in the presence of an intact U-rich IE1, a TIA-1 binding sequence. Co-transfection of TIA expression vectors did not restore splicing when the palindrome was mutated but did restore splicing when other sequences in the minigene construct were mutated, indicating a co-dependence of the IE1 and ESE for alt ex splicing. We did not experimentally define the molecular nature of this co-dependence, but several models can be considered. The model we favor is one in which both TIA binding to the IE1 and SRp55 and SRp20 binding to the ESE are required to recruit U1 snRNP complex to the 5' splice site (with the caveat that we have yet to show that SRp55 and SRp20 transactivate splicing of this exon) (Fig. 8). This model is based on the observations in splicing of Fas (13), MSL-2 (32), FGFR-2 (14), and TIA itself (19) that show that TIA binds to the U-rich intronic sequence located immediately downstream from a weak 5' splice site and promotes recruitment of U1 snRNP to the weak 5' splice site (18). Why both the ESE and IE1 are required is suggested by modeling of the secondary structure of the MYPT1 pre-mRNA (Fig. 8). The modeling suggested that this region forms a stem-loop with the splice site base-paired with the ESE. Thus, it would be proposed that trans-acting factors binding to the ESE are required to unmask the splice site and that without this TIA-1 cannot recruit U1 snRNP to the splice site. This model will require experimental testing. Alternatively, it is possible that TIA and SR binding to their cis-elements are cooperative in the intact MYPT1 pre-mRNA and that this is not evident when short RNA oligonucloetides are used to assay for protein binding. Finally, the assembly of splicing complexes involves the assembly of many proteins into a splicing complex, and the further identification of these proteins is required for a more complete understanding of the regulated splicing of this exon.

View larger version (28K): [in this window] [in a new window]   FIG. 8. Proposed model for TIA and SR protein regulation of splicing of MYPT1 central alt exon. Exons are shown as boxes, and introns are shown as horizontal lines connecting exons. A, top left, TIA binds to the U-rich IE1, and SRp55 and SRp20 bind to the ESE in tonic phenotype SMCs. These proteins may relieve the secondary structure that masks the splice site sequence and recruit the U1 to the weak 5' splice site, thereby promoting splicing. B, TIA and SR proteins fail to bind to the IE1 and ESE, respectively, possibly due to either low levels of expression of these proteins in phasic phenotype (posthatched gizzard) SMCs or/and expression of a tissue-specific suppressor protein binding to the ESS juxtaposed to the ESE and/or to the IE1. The alternative exon is skipped.

In summary, we have proposed a model in which cooperative action of TIA proteins and SRp55 and SRp20 are required to transactivate splicing of MYPT1 alt exon in smooth muscle and nonmuscle cells. Genetic depletion of TIA proteins causes significant reductions in splicing of the MYPT1 central alt exon. Splicing of the MYPT1 3' alternative exon and another smooth muscle/nonmuscle alternative exon is unaffected by TIA depletion. These results demonstrate the role of TIA and SR proteins in the regulated splicing of alternative exons and also highlight the incredible regulatory complexity in the generation of smooth muscle phenotypic diversity. Further research is required to identify how a multitude of positive and negative regulators of splicing interact to produce smooth muscle phenotypic diversity.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R01 HL-66171. 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.

^|| To whom correspondence should be addressed: BRB 422, 2109 Adelbert Rd., Cleveland, OH 44106-4958. Tel.: 216-368-0488; Fax: 216-368-0507; E-mail: saf9{at}po.cwru.edu.

¹ The abbreviations used are: SM, smooth muscle; SMC, smooth muscle cell; MYPT1, myosin phosphatase targeting subunit 1; TIA-1, T-cell inhibitor of apoptosis; TIAR, TIA-1-related; PTB, polypyrimidine tract-binding protein; MLC, myosin light chain; ESE, exonic splicing enhancer; SR, serine-arginine-rich; RT, reverse transcriptase; WT, wild type; WTE, whole tissue extract; EDn, embryonic day n; Dn, day n; nt, nucleotide(s); bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

	ACKNOWLEDGMENTS

 
We thank Dr. Nancy Kedersha for providing antibodies for TIA proteins; Dr. Hua Lou for providing expression plasmids for TIA-1, TIAR, and PTB; and Dr. Claudia Villabona for analysis of SR protein expression.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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