Human Molecular Genetics, 2002, Vol. 11, No. 5 577-587
© 2002 Oxford University Press
SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2ß1
Arizona State University, Department of Biology, Tempe, AZ 85287-1501, USA, 1Ottawa Health Research Institute, Ottawa, Ontario K1H 8L6, Canada and 2Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA
Received November 19, 2001; Revised and Accepted January 8, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is caused by the homozygous loss of survival motor neuron (SMN1). SMN2, a nearly identical copy gene, is present in all SMA patients; however this gene cannot provide protection from disease due to the aberrant splicing of a critical exon. SMN1-derived transcripts are exclusively full-length, whereas SMN2-derived transcripts predominantly lack SMN exon 7. A single non-polymorphic nucleotide difference (C in SMN1; T in SMN2) is responsible for the alternative splicing patterns. We have previously shown that transient expression of an SR-like splicing factor, hTra2ß1, stimulates inclusion of exon 7 in SMN2-derived mini-gene transcripts through an interaction with the AG-rich exonic splice enhancer within exon 7. We now demonstrate that a second splicing factor, SRp30c, can stimulate SMN exon 7-inclusion and that this activity required the same AG-rich enhancer as hTra2ß1. SRp30c did not directly associate with SMN exon 7; rather its association with the exonic enhancer was mediated by a direct interaction with hTra2ß1. In the absence of the hTra2ß1 binding site, SRp30c failed to complex with SMN exon 7. Taken together, these results identify SRp30c as a modulator of SMN exon 7-inclusion and provide insight into the molecular regulation of this critical exon.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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In humans, two nearly identical survival motor neuron genes (SMN1 and SMN2) exist on chromosome 5q13. However, it is the homozygous loss or mutation of SMN1 that is responsible for proximal spinal muscular atrophy (SMA) (1), the second most common autosomal recessive disease (2). The disease is characterized by loss of
-motor neurons in the spinal cord, which presents as proximal, symmetrical limb and trunk muscle weakness that ultimately leads to death. All three major forms of SMA, type I (severe), type II (intermediate) and type III (adult), are classified by age of onset and disease severity. The critical difference between SMN1 and SMN2 is a silent non-polymorphic C
T nucleotide transition in exon 7 (3,4). SMN1 contains a ‘C’ nucleotide at the +6 position of exon 7 and produces primarily full-length SMN transcripts, whereas SMN2 contains a ‘T’ nucleotide at the same position and produces low levels of full-length transcripts and high levels of a transcript that lacks exon 7. The latter transcript results in a truncated protein that is less stable and has a reduced ability to oligomerize (5,6), explaining why SMN2 cannot prevent SMA. However, family studies have clearly shown that SMN2 is a disease modifier in a dose dependent manner (7–9). The modifying effect of SMN2 has been confirmed in animals where it has recently been demonstrated that SMN2 transgenes are capable of rescuing the embryonic lethality of Smn–/– mice (9,10). Thus, the presence of intact SMN2 gene(s) in SMA patients provides a natural target for therapeutic intervention. Pre-mRNA processing is an essential step in eukaryotic gene expression that requires tremendous fidelity. The accurate identification of exon sequences, excision of intervening introns and ligation of exons is performed by several general splicing factors, including the ribonucleoprotein complexes U1, U2, U4/U6 and U5 snRNPs (11). SR and SR-like proteins perform additional regulatory roles in constitutive and alternative splicing (12). One likely role of SR proteins is to form a complex network of protein–protein and protein–RNA interactions that facilitates the recruitment of the splicing machinery and the assembly of competent splicing complexes. SR and SR-like proteins contain two highly conserved domains: an arginine/serine-rich (RS) domain and at least one RNA recognition motif (RRM). Typically the RS domain is regarded as the protein-interaction region (13,14), whereas the RRM is the RNA binding domain (15,16). RS domains are likely partially functionally redundant since these domains have been shown to be interchangeable in some systems (17). Recent studies have shown that splicing of particular pre-mRNAs can occur independently of RS domains (18). RNA binding sequences have been identified for several SR and SR-like proteins through SELEX procedures and in vivo splicing assays (19–22). Two general classes of binding motifs have been identified: a purine-rich enhancer consisting of GAR repeats (where R represents either G or A) (19,23–27) and CA-rich motifs, termed ACEs (24,28). Exonic splice enhancers (ESEs) stimulate the inclusion of the exons that the ESEs reside in, and exonic splice silencers (ESSs) suppress exon inclusion.
Understanding the molecular mechanisms that govern SMN exon 7 processing could lead to the development of therapeutic strategies for the treatment of SMA by suppressing the alternative splice of SMN2 exon 7. We have previously described an in vivo model system that was used to identify and characterize a cis-acting ESE sequence within SMN exon 7 (3–5) and an AG-rich ESE (SE2) is present within the central region of SMN exon 7 that is required for inclusion of exon 7 (5,29). hTra2ß1, an SR-like factor, associates with this enhancer to suppress exon 7-skipping and promote exon 7 inclusion (29). To further characterize the trans-factors required for SMN exon 7 inclusion, a panel of SR proteins was co-expressed with the SMN2 mini-gene. One factor was shown to alter SMN2 splicing patterns: SRp30c (30–32). SRp30c stimulated high levels of full-length expression from SMN2, although SRp30c bound directly to SMN exon 7 RNA inefficiently. SRp30c did efficiently form a trimeric complex with SMN exon 7 RNA in the presence of hTra2ß1, suggesting that this interaction is involved in SMN exon 7 inclusion. Taken together, these results identify SRp30c as a modifier of SMN exon 7 pre-mRNA splicing and demonstrate a direct interaction between two modifiers of SMN exon 7 splicing. Additionally, these results support a model in which multiple SR and SR-like factors are required for the identification and inclusion of this critical exon.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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SRp30c stimulates full-length expression from SMN2
We have previously developed a model system to dissect the molecular basis of SMN exon 7 processing (3–5,29). The SMN1 mini-gene exclusively produces full-length transcripts, while the SMN2 mini-gene produces lower levels of the full-length and high levels of the exon 7-skipped transcript (Figs 1A and 2). The full-length/exon skipped ratio from the SMN2 mini-gene has been established based upon numerous transfection experiments in various cell types (3–5,29). Consistently, full-length SMN is at 10–15% of the level of the exon-skipped product. This ratio served as an internal control for all experiments and was performed to ensure the RT–PCR analysis was consistent between various experiments. It should be noted that transient expression of SR proteins may lead to a decrease in total expression from mini-gene templates, but it is the relative ratio of exon skipped:full-length product within a single reaction that determines whether a factor modulates SMN exon 7 inclusion.
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To identify factors, in addition to hTra2ß1, that possessed a functional role in the inclusion of SMN exon 7, a previously cloned subset of SR proteins were co-transfected with the SMN2 mini-gene. Since the presence of large fusions might interfere with the activity of various SR proteins, expression vectors were selected based upon their small fusion tag or complete lack of additional fusion sequences. Co-transfection of SRp55, 9G8, SRp20, SRp40 and SC35 followed by RT–PCR analysis of plasmid-based transcripts did not stimulate full-length expression from the SMN2 mini-gene, nor did they dramatically increase the accumulation of the exon-skipped product (Fig. 1A). In contrast, SRp30c stimulated the inclusion of SMN exon 7, resulting in increased levels of the full-length product, and a concomitant decrease in the levels of the exon-skipped product (Fig. 1A). Finally, the SR factors did not affect expression from the SMN1 minigene (data not shown).
Since intracellular levels of SR factors can vary depending upon the cell type, several cell lines were examined to determine whether a particular cellular context affected the ability of SRp30c to stimulate full-length SMN expression. In two human cell lines, C33A and U20S (cervical carcinoma and osteosarcoma cell lines, respectively), SRp30c stimulated full-length expression from the SMN2 mini-gene, although less efficiently in C33A cells (Fig. 1B). Similar levels of SRp30c stimulation were observed in HeLa, COS and 293T cell lines (data not shown). In contrast, SRp30c did not stimulate full-length expression from the SMN2 mini-gene when co-transfected into a murine fibroblast line, A92L (Fig. 1B).
To further characterize the role of SRp30c in SMN exon 7 splicing, similar in vivo splicing assays were performed with SMN2 and expression vectors of either SRp30c or hTra2ß1. The ratio of full-length transcripts produced from SMN2 increased in a dose-dependent manner with increasing levels of the SRp30c expression vector, although similar levels of the hTra2ß1 expression vector were capable of inducing greater levels of SMN exon 7 inclusion (Fig. 2A). Co-transfection of 0.5 µg of hTra2ß1 vector stimulated high levels of full-length and a concomitant decrease of the exon 7-skipped product. This is consistent with previous studies in other cell types (4,29).
Titration experiments were performed to compare the effects of increasing amounts of hTra2ß1 and SRp30c on exon 7 inclusion. Increasing amounts of hTra2ß1 vector (10, 50, 150 and 500 ng) stimulated the production of SMN2-derived full-length transcript such that the majority of these transcripts were full-length (Fig. 2B). Increased levels of full-length stimulation could be observed with as little as 50 ng of the hTra2ß1 vector. Increasing amounts of SRp30c vector also stimulated increasing amounts of SMN2-derived full-length transcript such that exon-skipped levels were approximately equivalent to full-length levels. However, SRp30c was less efficient since 150 ng of the SRp30c vector was required to initially alter the relative ratio of exon-skipped:full-length (compare 50 ng of hTra2ß1vector) and at 500 ng of SRp30c vector, an approximate 1:1 ratio was observed (compared to approximately 9:1/FL:
7 for a similar amount of hTra2ß1 vector) (Fig. 2B). Levels greater than 500 ng of either vector did not further enhance full-length expression, nor could we detect an alteration in exon 7 inclusion when SRp30c and hTra2ß1 were co-expressed at various concentrations compared to when they were expressed individually (data not shown). In similar co-transfections assays, biomolecular interaction analysis (BIA) and western blotting with a monoclonal against the N-terminal epitope tag (haemagglutinin) present on SRp30c and hTra2ß1 demonstrated that similar levels of SRp30c and hTra2ß1 were expressed compared to when they were expressed individually (data not shown).
An AG-rich splice enhancer is required for SRp30c stimulation of full-length SMN
To determine the regions within SMN exon 7 pre-mRNA mediating stimulation of exon inclusion by SRp30c, in vivo splicing assays were performed with mutated SMN1 mini-genes that contained site-directed mutations within SMN exon 7 (Fig. 3). These experiments were performed in the SMN1 background since similar mutations in the SMN2 background result in a complete absence of detectable full-length expression (data not shown). SMN1 mini-genes containing single mutations within previously characterized exon 7 regions (SE1, SE2 and SE3) that are required for maximal levels of full-length expression (5,29) were transfected alone or with increasing amounts of SRp30c vector (0.25 and 0.50 µg). Disruptions of the SE1 or SE3 regions, putative CA-rich elements, were almost completely compensated by increasing amounts of SRp30c, resulting in high levels of full-length SMN and a decrease in the level of the exon-skipped product (Fig. 4A). In contrast, a disruption in the AG-rich SE2 element resulted in the exclusive production of the exon-skipped product. This mutation could not be overcome even at the higher level of SRp30c vector (Fig. 4A, lane 6).
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To further characterize the requirement of the SE2 region in stimulation of SMN exon 7 inclusion by SRp30c, expression was analyzed from SMN1 templates containing double SE mutations: SE1 and SE2, SE1 and SE3, or SE2 and SE3. Even at the highest concentration of SRp30c, full-length expression was not detected from SMN1 templates with the SE1/2 and SE2/3 mutations (Fig. 4B). In contrast, SRp30c could stimulate full-length expression from the SE1/3 double mutation, although full-length activation was reduced compared to the ability of SRp30c to stimulate expression from templates with either SE1 or SE3 mutations singly.
To determine which region of the SE2 element is required for SRp30 activation, smaller mutations within the SE2 element (Fig. 3) were analyzed for their effects upon SRp30c-mediated stimulation of full-length expression. Disruption of the 5'-most (SE2a) and central region (SE2b) had a modest effect upon SMN splicing. However, both mutations could be overcome to near wild-type levels by the addition of SRp30c vector as determined by comparing the full-length to exon-skipped ratio (Fig. 4C). In contrast, SRp30c could not compensate for a disruption affecting the 3' segment of the SE2 element, SE2c (Fig. 4C). These results demonstrate that an intact SE2 element is required for the ability of SRp30c to efficiently stimulate full-length SMN expression.
Indirect association of SRp30c with SMN exon 7 is mediated by hTra2ß1
To determine whether SRp30c associated with SMN exon 7 RNA, immunopurified haemagglutinin (HA)-tagged SRp30c was incubated with (Fig. 5, lanes 2, 5 and 8) or without (Fig. 5, lanes 1, 4 and 6) in vitro splicing competent HeLa nuclear extract with various 32P-labeled RNA templates corresponding to SMN exon 7 sequences. RNA/protein complexes were UV cross-linked, immunoprecipitated with an anti-HA monoclonal antibody, washed extensively and bound fractions were blotted on filter paper and analyzed by scintillation counting (Fig. 5). In this assay, low levels of SMN exon 7 RNA were associated with SRp30c compared to background levels (Fig. 5 lanes 1 and 4). However, increased levels of the SRp30c/RNA complex were detected following the addition of in vitro splicing competent HeLa nuclear extracts to immunopurified SRp30c (Fig 5, lanes 2 and 5). This association was SE2-dependent as evidenced by the SE2-mutant RNA template that did not support SRp30c/RNA complex formation (Fig. 5, lanes 7 and 8). These results suggest that efficient association of SMN exon 7 by SRp30c occurs in the presence of additional factors present within in vitro splicing competent nuclear extracts. As expected, untransfected extracts incubated with in vitro splicing extracts did not associate with appreciable levels of the RNA templates (Fig. 5, lanes 3, 6 and 9).
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A potential candidate for mediating association of SRp30c with SMN exon 7 RNA is hTra2ß1. This is based on three observations: (i) hTra2ß1 binds directly to the AG-rich splice enhancer; (ii) SRp30c and hTra2ß1 require the same enhancer region (SE2) to stimulate exon 7 inclusion; and (iii) in a yeast two-hybrid system, a positive interaction between SRp30c and hTra2ß1 has been reported (33). BIA studies were performed to characterize the direct in vitro binding of SRp30c to hTra2ß1. Recombinant hTra2ß1 immobilized on a BIA sensor chip specifically captured recombinant SRp30c (Fig. 6A) and this complex was still detectable, although at reduced levels, in salt concentrations up to 300 mM NaCl (Fig. 6B). Similar binding experiments were performed in the absence of hTra2ß1, demonstrating that SRp30c did not bind directly to the chip or the capturing antibody (Fig. 6A), confirming the specificity of the interaction.
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BIA was used to determine whether an RNA/SRp30c/hTra2ß1 complex formed in vitro. Biotinylated wild-type SMN exon 7 RNA was immobilized on a streptavidin sensor chip. When SRp30c was incubated with SMN exon 7 RNA in the absence of recombinant hTra2ß1, SRp30c bound inefficiently to the RNA substrate (Fig. 7A). When SMN exon 7 RNA was pre-bound by recombinant hTra2ß1, levels of SRp30c binding were increased nearly 7–8-fold (Fig. 7A). As expected, binding by hTra2ß1 to SMN exon 7 RNA was dependent upon the AG-rich SE2 element. Three wild-type copies of SE2 in tandem (3xSE2) captured even greater levels of hTra2ß1, whereas three wild-type copies of SE1 (3xSE1), a CA-rich region within SMN exon 7, were bound poorly by hTra2ß1 (Fig. 7B). A similar dependence upon the SE2 region in hTra2ß1 binding was observed when binding experiments were performed with increasing salt concentrations (Fig. 7B).
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To further characterize the SRp30c/hTra2ß1/RNA trimeric complex, binding studies were performed on three templates: SMN exon 7, 3xSE2 and 3xSE1. SRp30c failed to efficiently associate with any of the RNA templates in the absence of hTra2ß1 at any salt concentration (Fig. 7C, black bars and data not shown); however, pre-binding of hTra2ß1 to 3xSE2 and wild-type SMN exon 7 increased the levels of associated SRp30c by
10- and 5-fold, respectively (Fig. 7C, compare 3xSE2 ± hTra2ß1; Fig. 7A). The data presented reflect only the association of SRp30c with the RNA/hTra2ß1 complex, not the pre-binding of hTra2ß1 to the various RNA substrates. When the 3xSE1 template that did not support hTra2ß1 binding was pre-treated with hTra2ß1, efficient SRp30c binding was no not detected (Fig. 7C). Pre-binding of hTra2ß1 was performed at low salt concentrations (150 mM NaCl) and allowed hTra2ß1 to be stably associated with the RNA templates. Increasing salt concentrations in the SRp30c injected samples did not cause dissociation of hTra2ß1 from the RNA templates (data not shown). Taken together these results demonstrate that that ability of SRp30c to stimulate SMN exon 7 inclusion requires the SE2 region and this activity is mediated, at least in part, by a direct interaction with hTra2ß1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Although SMN2 encodes an identical protein and shares 99% identity with SMN1 at the nucleotide level, it cannot provide protection from disease development in the absence of SMN1. The reason why SMN2 cannot functionally compensate for the loss of SMN1 is the dramatic reduction in SMN2-derived full-length transcript due to the high level of SMN exon 7 skipping (1,3,4,34). A single nucleotide difference 6 nucleotides downstream from the 5' end of SMN exon 7 (C in SMN1; T in SMN2) causes the differences in splicing patterns, although the underlying mechanism disrupted by this single nucleotide change is unknown (3,4). In this report, we identified SRp30c as a factor capable of compensating for the C/T transition by stimulating high levels of SMN2-derived full-length SMN transcript. SRp30c is a member of the SR class of splicing regulatory factors although its mode of action has not been determined (30–32). SRp30c was found to associate with SMN exon 7 in a trimeric complex through a direct interaction with hTra2ß1. We have previously demonstrated that hTra2ß1 interacts with SE2, the AG-rich enhancer sequence located in the center of SMN exon 7. Consistent with this, an RNA template with three copies of the SE2 element significantly increased hTra2ß1 and consequently SRp30c captured on a sensor chip, whereas removal of the SE2 sequence abolished binding of both proteins. The ability to bind to this region by these two factors correlates with their abilities to activate SMN exon 7 inclusion since mutations within SE2 that could not be compensated by the over-expression of SRp30c also did not support hTra2ß1/SRp30c complex formation (29).
These findings suggest that SRp30c and hTra2ß1 may function together to compensate for the C/T exchange in SMN2, although transient co-expression of both factors failed to promote synergistic or additive increases in exon 7 inclusion (data not shown). Potentially, the inability to further stimulate SMN exon 7 inclusion is due to endogenous levels of a rate-limiting splicing factor essential for efficient SRp30c/hTra2ß1 function. Although several hTra2ß1-interacting factors are known (29,35–37), relatively little is known regarding the interaction of SRp30c with other splicing factors (30,31), and additional intermediates may further strengthen the association of SRp30c with SMN exon 7. Further experiments are needed to identify additional splicing factors that are incorporated into the RNA/SRp30c/hTra2ß1 complex.
In the previous analysis of SR and SR-like factors involved in SMN exon 7 splicing, large N-terminal GFP fusions were expressed with the splicing factors (29). A large fusion could disrupt important protein interactions since a primary function of SR and SR-like factors is likely to facilitate binding of additional splicing factors through a network of protein interactions. Furthermore, a recent study indicated that N-terminal fusions interfered with SF2/ASF activity (18). Therefore, in this study either the N-terminal fusions were absent or they were reduced to a small number of amino acids. This difference likely explains why SRp30c was previously shown not to have an effect upon SMN exon 7 splicing, whereas in this report, SRp30c was capable of stimulating full-length expression.
Recent work strongly suggests that many point mutations that alter exon inclusion efficiencies can be attributed to a disruption of trans-factor binding sequences (38). Disruption of enhancer function correlates with the ability of the enhancer to be bound by specific SR or SR-like factors. Potentially, the SMN exon 7 C/T transition disrupts a splice enhancer region, resulting in SMN exon 7 skipping. However, by over-expressing factors with recognition motifs outside of the C/T region, full-length expression can be restored. Although it appears unlikely that SRp30c interacts directly with the C/T region, it remains possible that this region helps stabilize the SRp30c/hTra2ß1 interaction. Although additional putative ESEs, SE1 and SE3, have been identified within SMN exon 7 which more closely resemble CA-rich motifs (ACEs), these elements were not required for SRp30c function since mutation of SE1 and SE3 could be overcome by co-transfection of SRp30c. While these sequences were required for maximal levels of full-length expression from SMN genes, they were dispensable for SRp30c-mediated activation and likely do not comprise an SRp30c functional motif.
Essentially all SMA patients retain at least a single copy of SMN2. Therefore, stimulating exon 7 inclusion of SMN2-derived transcripts is a viable therapeutic approach. The identification of SRp30c as a modulating factor of SMN exon 7 inclusion addresses the molecular regulatory mechanism of this critical exon. While relatively high levels of endogenous SRp30c and the lack of target specificity argue against SRp30c as a therapy for SMA, SRp30c can play an important role in the development of vectors designed to identify compounds that pharmacologically convert SMN2 splicing to that of SMN1. Furthermore, as compounds are identified that convert SMN2 splicing patterns, it will be critical to understand the molecular signals that lead to exon 7 inclusion and to dissect the downstream molecular targets of these compounds.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Mini-genes
Mini-genes containing wild-type SMN1, SMN2 and mutations have been described previously (4,5,29). Briefly, genomic SMN sequences were amplified to include SMN exon 6 and the 5'-proximal 462 nucleotides of SMN exon 8. Mutations disrupt CA-rich (SE1, SE3) and AG-rich (SE2) elements within SMN exon 7 (5) as indicated (Fig. 3) with ‘T’ substitutions in the DNA sequence, but do not introduce premature termination codons into the SMN reading frame to avoid exon 7 skipping due to potential reading frame-dependent mechanisms.
Expression vectors
Vectors for the subset of SR factors (SRp20, SRp30c, SC35, SRp40, SRp55 and 9G8) were a generous gift from Adrian Krainer (Cold Spring Harbor Laboratory). The hTra2ß1 and SRp30c cDNAs were amplified (hTra2ß1: forward, 5'-CCG CGT GGA TCC TGA AGC GAC AG-3'; reverse, 5'- CGA TCT CGA GTT AAT AGC GAC-3'; SRp30c: forward, 5'-GAT CGG ATC CAG ATG TCG GGC TGG GCG GAC GAG CG-3'; reverse, 5'-GAT CCT CGA TCA GTA GGG CCT GAA AGG AGA G-3') from previously described vectors (29,32) and cloned between the BamHI and XhoI sites in pcDNA3 (Invitrogen) that contains an N-terminal HA-tag (6).
In vivo splicing assays
In vivo splicing assays were performed as described previously (4,5,29), but with minor modifications. Approximately 1 x 105 cells per 60 mm plate were transfected with 1.0 µg of mini-gene template and the indicated amount of SR expression vector (10, 50, 150 or 500 ng, as indicated) using Lipofectamine (8 µl) and PLUS Reagent (6 µl) (Invitrogen/Life Technologies). Total RNA was isolated 36 h post-transfection using Trizol (Invitrogen/Life Technologies). First strand cDNA synthesis was performed with 1 µg total RNA, 1.5 µl oligo dT (0.5 mg/ml), 1x first strand buffer, 100 mM DTT, 200 µM dNTPs and 50 U Super Script II (Invitrogen/Life Technologies) at 42°C/50 min followed by 70°C/10 min. To ensure amplification of plasmid-derived transcripts, PCR primers that annealed specifically to a portion of the plasmid backbone common to all mini-gene transcripts were used in the subsequent PCR amplification [94°C/1.5 min; (90°C/1 min; 58°C/1 min; 72°C/2 min) x15, 20 or 25] (4,5). An internal control amplification of the hypoxanthine phosphoribosyl-transferase gene was performed in initial experiments to ensure linear amplification. Products were either resolved on a 6% sequencing gel (Fig. 1A) or a 2% agarose gel (Figs 1B, 2A and B and 4A–C). Similar PCR reactions were performed with 15 (Fig. 2B) and 20 cycles and resolved by a 6% sequencing gel and quantitated by PhophorImager (BioRad) analysis to demonstrate linear amplification (data not shown) (4,5).
In vitro RNA binding
In Figure 5, sense strand RNA corresponding to exon 7 from SMN1, three wild-type copies of SE2 (3xSE2), or exon 7 from SMN1SE2 was transcribed in vitro and uniformly labeled with all four [-32P] NTPs (NEN). RNAs were incubated on ice with HA-immunopurified fractions from cellular extracts from 2 x 60 mm dishes transfected with 1.0 µg HA-SRp30c. When indicated (lanes 2, 3, 5, 6, 8 and 9) HA-SRp30c fractions were incubated with 30 µg of in vitro splicing competent HeLa nuclear extract with 1x splicing buffer for 15 min before the addition of heparin (final concentration 2 mg/ml). Samples were UV-irradiated (480 000 µJ/cm2) and HA-bound fractions were collected with Protein A/G Sepharose (Invitrogen/Life Technologies). SRp30c-associated 32P-labeled RNA was determined by scintillation counting bound fractions. Radioactive counts from SMN1 exon 7 with immunopurified HA-SRp30c served as the relative baseline (set at a value of 1.0). The relative radioactive counts from the other binding experiments were expressed compared to the raw value obtained in lane 1. Experiments were performed in duplicate and representative results are shown.
Biomolecular interaction analysis
BIA was performed as previously described, but with minor modifications (39). For direct SRp30c/hTra2ß binding, a Cm 5 sensor chip with amine-coupled rabbit anti-mouse IgG antibody was used. Recombinant hTra2ß1 was captured with an anti-thioredoxin monoclonal antibody (MANTRX1) that recognized the N-terminal tag from the pRSET-C expression vector and then probed with recombinant poly-histidine SRp30c (6xHis–SRp30c). The sensor chip coupled to the antibody was initially probed with SRp30c in the absence of hTra2ß1 to show SRp30c did not interact directly with either the chip or the monoclonal antibody. To demonstrate that hTra2ß1 specifically captured SRp30c, a mock bacterial extract was used to probe the hTra2ß1 sample. The large vertical spikes are a consequence of a low level of urea present in the binding buffer used in the extraction procedure.
For RNA binding studies, a streptavidin (SA) chip was used to capture biotinylated sense strand RNA corresponding to exon 7 from SMN1, three wild-type copies of SE2 (3xSE2) (5'-CTA GAC AAA AGA AGG AAG GCA AAA AGA AGG AAG GCA AAA AGA AGG AAG GAA-3'), or three wild-type copies of SE1 (3xSE1) (5'-CTA GAG GTT TCA GAC AAA AGG TTT CAG ACA AAA GGT TTC AGA CAA AA-3'). Purified recombinant hTra2ß1 was used in BIA at 150 mM or increasing salt concentrations (50, 150, 300, 500 and 750 mM NaCl). To demonstrate that hTra2ß1 facilitates the indirect association of SRp30c with SMN exon 7 RNA, captured RNA was probed with SRp30c before and after the addition of hTra2ß1. The experiment was repeated in increasing concentrations of NaCl to show the relative strength of binding. Protein concentrations were quantitated prior to injections and confirmed by Coomassie staining (data not shown).
Recombinant protein purification
SRp30c and hTra2ß1 were expressed from pRSETc (Invitrogen) and pET32c (Novagen), respectively. Transformed bacteria were induced with 1 mM IPTG for 16 h at 37°C. Expressed fusion protein was purified from inclusion bodies by sequential extraction with increasing concentrations (2, 4, 6 and 8 M) of urea in phosphate-buffered saline. Extracted protein was further purified by metal chelate affinity chromatography using Histidine Binding Resin (Novagen) in the absence of urea.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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We thank Adrian Krainer for SR protein expression vectors, and Glenn Morris for SMN and thioredoxin antibodies and BIAcore access. P.J.Y. was supported by a post-doctoral fellowship from Families of SMA. This work was funded by grants from the Muscular Dystrophy Association (C.L.L. and E.J.A.) and the National Institutes of Health (C.L.L., RO1 NS41584-01; E.J.A., RO1 NS40275).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 480 965 5444; Fax: +1 480 965 2519; Email: clorson@asu.edu
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REFERENCES |
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