Human Molecular Genetics, 2000, Vol. 9, No. 2 259-265
© 2000 Oxford University Press
An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN
1Department of Dermatology, New England Medical Center and Tufts University School of Medicine, Boston, MA 02111, USA and 2Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA
Received 15 September 1999; Revised and Accepted 5 November 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The survival motor neuron genes, SMN1 and SMN2, encode identical proteins; however, only homo- zygous loss of SMN1 correlates with the development of spinal muscular atrophy (SMA). We have previously shown that a single non-polymorphic nucleotide difference in SMN exon 7 dramatically affects SMN mRNA processing. SMN1 primarily produces a full-length RNA whereas SMN2 expresses dramatically reduced full-length RNA and abundant levels of an aberrantly spliced transcript lacking exon 7. The importance of proper exon 7 processing has been underscored by the identification of several mutations within splice sites adjacent to exon 7. Here we show that an AG-rich exonic splice enhancer (ESE) in the center of SMN exon 7 is required for inclusion of exon 7. This region functioned as an ESE in a heterologous context, supporting efficient in vitro splicing of the Drosophila double-sex gene. Finally, the protein encoded by the exon-skipping event,
7, was less stable than full-length SMN, providing additional evidence of why SMN2 fails to compensate for the loss of SMN1 and leads to the development of SMA. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal muscular atrophy (SMA) is a common autosomal recessive disease characterized by degeneration of
-motor neurons. Two nearly identical survival of motor neuron (SMN) genes are typically present on chromosome 5q13: SMN1 and SMN2 (1,2). Only homozygous loss of SMN1 correlates with SMA development (1). These two SMN genes are >99% identical at the nucleotide level and encode SMN (1,3), a 294 amino acid RNA-binding protein (4). SMN has been shown to play a role in the biogenesis of small ribonuclear proteins (snRNPs) through association with SMN-interacting protein 1 (SIP-1) and Sm proteins (5,6), and enhances in vitro splicing, potentially as a regenerative factor for the general splicing machinery (7).
We have shown that development of SMA correlates with aberrant splicing of SMN exon 7 (8). SMN1 mRNA predominantly expresses a full-length transcript whereas SMN2 produces low levels of full-length RNA and an aberrantly spliced product lacking exon 7, termed 7 (1,9). SMN7 protein, predicted to be slightly smaller than full-length SMN, has not been detected by western analysis, and has therefore been presumed to be less stable. We have reported that a single nucleotide difference between SMN1 and SMN2 within exon 7 (exon 7+6, where +6 refers to the sixth nucleotide in SMN exon 7) is responsible for their splicing patterns and the subsequent development of SMA (8). Proper mRNA processing of this exon is critical since several mutations in the splice sites adjacent to exon 7 have been isolated from SMA patients and SMA hybrid genes exhibit high levels of aberrant mRNA processing in this region (1,10). Little is known concerning the processing of SMN exon 7 or the functional significance of the resultant 7 protein in the development of SMA. The exon 7+6 nucleotide was predicted to have disrupted an exonic splicing enhancer (ESE); however, this activity has yet to be demonstrated. Elucidating the processing mechanism(s) regulating SMN full-length expression relative to the exon-skipped mRNA is an important step in furthering the molecular pathogenesis of SMA.
Pre-mRNA processing is a critical step in eukaryotic gene expression (11,12). The accurate identification and excision of intron sequences and the subsequent ligation of 5' and 3' splice sites is carried out by a large complex consisting of U1, U2, U4/U6 and U5 snRNPs and a group of RNA-binding factors termed SR proteins (13,14). SR proteins typically contain two critical regions: an arginine/serine-rich (RS) domain (13,14) and at least one RNA recognition motif (RRM), which function to mediate protein–protein interactions and RNA binding, respectively (15,16). Processing of constitutive and regulated pre-mRNAs requires SR proteins (17–19). ESEs are found in constitutive and regulated exons and serve as binding sites for SR proteins. Two general classes of binding motif have been identified: a purine-rich enhancer consisting of GAR repeats (20–25) (where R represents either G or A) and CA-rich motifs, termed ACEs (22,26). Through a series of protein–protein interactions, SR factors bound to ESEs are generally believed to function by recruiting the splicing machinery and promoting the cooperative assembly of competent splicing complexes (17,22–24,27,28). Another function of ESEs is to compensate for suboptimal 3' splice sites, thereby promoting inclusion of an otherwise poorly defined exon (29,30). Potentially, one or more ESEs are operative in the constitutive inclusion of SMN exon 7. In this report, we sought to identify and characterize the cis-acting elements responsible for SMN exon 7 processing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Two nearly identical SMN genes are typically present in normal unaffected individuals; however, only the presence of SMN1 and the production of high levels of full-length SMN protein protect against the development of SMA. A single nucleotide difference between SMN1 and SMN2 affects the generation of mRNA species (3,8). The SMN1-derived nucleotide (exon 7+6/C) results in the production of full-length SMN, and the SMN2-derived nucleotide (a C
T transition) (Fig. 1A) results in an aberrantly spliced product lacking exon 7, called 7. The following experiments were designed to identify and characterize the regulatory mRNA-processing elements surrounding SMN exon 7. A semi-quantitative radiolabeled polymerase chain reaction (PCR)-based assay was developed to analyze the effects of the SMN exon 7 mutagenesis. pSMN1 and pSMN2 plasmids contain genomic sequences from SMN exons 6–8 and have previously been shown to recapitulate the splicing patterns of the respective endogenous SMN genes (8). Following transient transfection, total RNA was isolated and used to generate cDNA. PCR amplification utilized a primer set specific to the plasmid segment flanking the SMN sequences; therefore, only plasmid-derived SMN transcripts were detected. This was necessary since all cell lines contain endogenous SMN transcripts. Following 15 rounds of amplification, detectable pSMN1 expression was exclusively detected as a full-length cDNA product that included exons 6, 7 and 8, whereas pSMN2 expression was predominantly the exon-skipped product, 7 (Fig. 1B). These results have been repeated numerous times and the quantitative relative ratios of full-length:7 for pSMN1 and pSMN2 are routinely >99 ± 1 and 16 ± 3%, respectively. Control reactions that reflected the typical full-length:7 ratios were performed with these templates in each of the following experiments to maintain consistency throughout (data not shown).
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It was critical to demonstrate that the PCR results represented linear amplification and that accurate amplification of both mRNA species in a single reaction occurred. To this end, full-length and
7 cDNAs derived from the pSMN2 mini-gene were cloned into the same vector used for genomic expression. Therefore, the amplification products from the following control PCR reactions were identically sized as the spliced products from pSMN1 and pSMN2. The same primer set was then used in a series of competitive PCR reactions utilizing known concentrations of cDNA-containing plasmids. Using 15 PCR cycles, representative amplification occurred over two orders of magnitude in template concentration (Fig. 1C). Similar results were obtained with up to 20 cycles (data not shown). Therefore, this assay could be used for the analysis of SMN exon 7 mRNA processing requirements.
To characterize SMN exon 7 mRNA processing, we sought to determine whether the critical SMN exon 7+6 position absolutely required the SMN1-derived C nucleotide. Previously, we reported that the CT (U in RNA sequence) transition within SMN exon 7 resulted in the abundant accumulation of the exon-skipped mRNA species, 7. Although referred to throughout this proposal as a T nucleotide, it is implied that T refers to the DNA sequence and will transcribe into a U residue in RNA sequence. Site-specific mutagenesis introduced either an A or G substitution for this C nucleotide. In the nomenclature for the following mutations, the parent plasmid is indicated and the mutation follows the dash. Introduction of an A nucleotide resulted in a low level of exon skipping, whereas the G substitution had an even lower, though detectable, level of exon-skipped product (Fig. 2A). Interestingly, ESEs are typically sensitive to U substitutions in the enhancer regions, whereas C and G residues typically have a neutral effect (24,25,31), suggesting that SMN exon 7 may contain one or more ESEs.
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ESEs are cis-elements that serve as binding substrates for splicing factors that are recruited to exons, and in turn regulate mRNA processing in addition to the typical 5' and 3' splice signals. The T at exon 7+6 in SMN2 might disrupt such an ESE. We therefore determined whether SMN exon 7 contained splice enhancers. Three potential enhancer regions were identified by sequence (20–25). Putative splice enhancer region 1 (SE1) is a 5' CA-rich region, SE2 is a centrally located AG-rich element and SE3 contains CA sequences in the 3' end of the exon (Fig. 1A). SE2 would be predicted to represent the strongest putative enhancer element, since uninterrupted AG-rich stretches have previously been shown to function as potent ESEs (22–25). The three potential splicing enhancer motifs were disrupted by introducing mutations into an otherwise wild-type pSMN1 background. In this strategy, each mutated nucleotide would result in a U residue in the RNA sequence. The mutations are indicated above each nucleotide within the putative elements (Fig. 1A). Importantly, none of these mutations introduced a premature termination codon (PTC), since PTCs can affect exon retention (32). In contrast to the parent construct pSMN1, which expressed undetectable levels of the exon-skipped product, the introduction of SE1 or SE3 mutations resulted in the accumulation of full-length and
7 species to similar levels (Fig. 2B, lanes 3 and 5). The SE2 mutation dramatically altered splicing patterns, yielding abundant 7 and undetectable levels of full-length transcripts (Fig. 2B, lane 4). The combination of either SE1 or SE3 mutations in conjunction with SE2 resulted in expression levels similar to those observed with SE2 alone (Fig. 2B, lanes 6 and 8), i.e. exclusively 7 transcripts. Introduction of both SE1 and SE3 mutations into pSMN1 further increased the amount of 7 relative to full-length compared with either mutation individually; nonetheless, a low level of full-length product was still readily detectable (Fig. 2B, lane 7). Taken together, these results suggest that the AG-rich SE2 region functions as an ESE within a constitutively spliced exon.
One function of ESEs is to strengthen suboptimal splice signals flanking the exon (11,30). Although the 3' and 5' splice sites flanking SMN exon 7 are near consensus, the polypyrimidine tract (PPT) could be suboptimal since a non-consensus A residue interrupts the pyrimidine stretch (29,30). In the following mutations, the relative strength of the SMN exon 7 PPT was increased by replacing the exon 7 PPT with 12 consecutive U residues (Fig. 1A). The uninterrupted stretch of 12 U residues in the RNA abrogated the typical exon-skipped pattern of pSMN2-derived transcripts and resulted in high levels of full-length transcript and no 7 (Fig. 2C, lanes 1 and 2). The enhanced PPT (12U PPT) also restored full-length expression to pSMN1 constructs with either SE1 or SE3 mutations (Fig. 2C, lanes 4 and 6). However, no increase in full-length mRNA was detected when the exon 7 RNA contained the SE2 mutation (Fig. 2C, lane 5). Furthermore, exon 7 inclusion was not increased by the 12U PPT within the context of SE1/2 or SE2/3 double mutations, whereas the SE1/3 double mutation was partially compensated by the increased strength of the PPT (Fig. 2C, lanes 7–9).
These studies highlight the importance of the SE2 element in SMN exon 7 processing. To characterize this region further, a series of smaller sequential substitution mutations were introduced into the AG-rich region. These mutations replaced three consecutive bases from the AG-rich region with three U residues, dividing SE2 into three subdomains: SE2a, SE2b and SE2c (Fig. 1A). Disruption of the first two SE2 regions, SE2a and SE2b, with UUU substitutions slightly increased the relative amount of the exon-skipped product. Mutation of SE2c resulted in a concomitant decrease of full-length and an increase of 7 mRNA (Fig. 3, lanes 4–6). Finally, the entire region spanning SE2 was replaced with its complementary sequence. This severe mutation also resulted in high levels of 7 expression (Fig. 3, lane 3). Taken together, these results demonstrate that the AG-rich SE2 ESE is necessary for constitutive inclusion of SMN exon 7. This ESE is also present in SMN2; however, it fails to overcome the T at position +6 that causes exon 7 skipping.
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The AG-rich element in SMN exon 7 functions in this experimental context to facilitate high levels of constitutive full-length expression, but it is generally acknowledged that a true ESE should function in a heterologous context. To demonstrate conclusively that the AG-rich region of SMN exon 7 functioned as an ESE, we tested the ability of the SE2 region to support efficient splicing of the Drosophila double-sex (dsx) gene (17,24). Using the dsx cassette, which has been used previously to characterize a number of ESEs (17,25), the complete sequence of SMN1 or SMN2 exon 7 was cloned downstream of dsx exon 4 (Fig. 4A). Additionally, three or five wild-type copies of the AG-rich region identified by the SE2 mutation were similarly positioned downstream of dsx. All four in vitro splicing templates contained fusion exons of essentially the same size, and therefore produced similarly sized spliced products. Relatively low levels of properly spliced product were detected with either the dsx-SMN1 or -SMN2 templates (Fig. 4B, lanes 1 and 2). Insertion of the three copies of the AG-rich element had a modest effect on splicing efficiency; however, five copies of wild-type SE2 sequences supported high-efficiency in vitro splicing (Fig. 4B, lanes 3 and 4). Similar results were obtained following a 30 or 60 min reaction time (data not shown). The positive control contains multiple strong ESEs from the bovine papillomavirus (BPV) that result in similar sized products (Fig. 4B, lane 5) (20–25). The AG-rich region within SMN exon 7 therefore functions as an ESE within its natural context in vivo and in a heterologous in vitro splicing system.
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Our data show that the aberrant splicing of SMN2 results in expression of SMN
7, a protein that cannot functionally compensate for the loss of full-length protein in SMA. Having identified a critical genetic element that regulates the expression of full-length versus 7, we addressed the significance of the aberrant splice in the development of SMA and SMN protein biochemistry. Whereas the 7 protein self-associates less efficiently and fails to enhance in vitro splicing, there has been speculation that 7 is less stable than full-length protein, and therefore is unable to provide protection from development of disease. To compare the intracellular stability of the SMN proteins, N-terminal hemagglutinin (HA) epitope-tagged HA:SMN or HA:7 SMN expression constructs were transiently expressed in U2OS cells, a human osteosarcoma cell line. Expression was under the control of the constitutively expressing cytomegolovirus immediate-early promoter. Twenty-four hours post-transfection, cells were washed extensively and treated with 10 mM cycloheximide to inhibit protein synthesis. Cells were harvested at time zero and five subsequent time points (T0 = 0; T1 = 45; T2 = 90; T3 = 120; T4 = 150; T5 = 180 min). Equivalent amounts of cellular extract were analyzed by western blot using HA monoclonal antibody. Full-length HA:SMN remained largely present even at the final time point, 180 min (Fig. 5, lanes 2–7), whereas 7 degradation was readily apparent (Fig. 5, lanes 8–13). Although full-length HA:SMN was more abundant at the first time point compared with HA:SMN7, the difference in stability can still be observed since at the last time point only very low levels of SMN7 were present. The membrane was stained to confirm that loading and transfer were equivalent (data not shown). Taken together, these results identify a critical genetic element required for the constitutive inclusion of SMN exon 7. The subsequent protein product of the exon-skipping event, which is the principal product of the SMN2 genes, was less stable and therefore provides further evidence of why SMN7 protein fails to protect against the development of SMA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification and characterization of a purine-rich enhancer within the constitutively spliced SMN exon 7
One of the most critical aspects of SMA pathogenesis is understanding why SMN2 cannot protect against SMA in the absence of the nearly identical SMN1 gene. SMA patients are clinically categorized based on age of onset and severity of symptoms: type I (severe) to type III (mild) (33). Although not protective against the development of SMA, SMN2 acts as a disease modifier and decreases SMA severity in a dose-dependent manner. There is an excellent correlation between higher number of SMN2 copies and milder forms of SMA (34). Several groups have demonstrated that the steady-state levels of full-length, wild-type SMN protein correlated with disease development (35,36). Thus, the low levels of SMN2-derived full-length SMN protein are likely to provide partial protection from disease. Furthermore, the primary product of SMN2, SMN
7 protein, is defective for self-association (37), stimulation of in vitro splicing (7) and, as we show here, is much less stable. Our study was designed to identify and characterize the mRNA processing elements regulating SMN exon 7 splicing events. An AG-rich enhancer in the center of exon 7 was necessary for constitutive inclusion of SMN exon 7 in vivo and was sufficient for stimulating efficient in vitro splicing. The mutational analysis of SE1 and SE3 identified additional requirements for maximal levels of exon inclusion and suggested that exon 7 contains multiple layers of regulation. Interestingly, the effects of the SE1 and SE3 mutations were abrogated by increasing the relative strength of the upstream PPT. In contrast, the SE2 region was essential and disruption of this region could not be overcome by increasing the PPT strength. Notably, the AG-rich motif within SE2 was the closest of the three elements to a consensus ESE motif (13,29,38). These results are consistent with the exon 7 AG-rich element functioning as an ESE. ESEs have been identified within constitutively and alternatively regulated exons, and one mechanistic role of ESEs is to compensate for suboptimal flanking splice signals. RNA stability is not likely to be contributing to these results since several mutations that reduce full-length expression were retained in the final transcript by increasing the strength of the PPT. In further support of SE2 acting as a bona fide ESE, SE2 functioned in a heterologous context by stimulating splicing of the dsx splicing cassette. Interestingly, the AG-rich motif is extremely well conserved across divergent species in which SMN has been identified.
Proper splicing of SMN exon 7 is likely to involve multiple regulatory elements. It is possible that the critical C/T nucleotide difference in exon 7 between SMN1 and SMN2 does not disrupt the AG-rich SE2. The small effect of the CA and CG substitutions (Fig. 2A) supports the notion that an enhancer has been disrupted. Previously characterized ESEs are particularly sensitive to U residues, less so to As, and Gs can be neutral (24,25,31). This pattern of nucleotide preference was closely adhered to at the exon 7+6 position. Relatively little is known, however, about the role of the 5'-most exonic sequences regarding splice site selection, and the C/T transition may represent a disruption at a different step of the splicing reaction. A single nucleotide substitution flanking the +6 position, however, did not affect exon 7 processing (data not shown). Since little sequence homology exists between heterologous 5' exon sequences, the mechanism underlying why the +6 T causes exon skipping is currently being investigated.
The in vitro splicing results confirm that the SE2 region acts as an ESE. However, in the context of a single copy of full-length exon 7, SMN1 or SMN2 in vitro splicing was inefficient and the difference between SMN1 and SMN2 observed in vivo was not apparent. Heterologous ESEs have been identified using similar approaches, and it is not uncommon to require multiple copies of ESEs to mediate efficient splicing in vitro, likely due to the absence of additional elements that serve to complement the enhancer present in its natural context. Several possibilities exist as to why a single SMN1 or SMN2 chimeric exon 7 template did not recapitulate their respective in vivo splicing patterns. The distance from the 3' splice site may influence the activity. The SMN sequences are fused downstream of the dsx exon 4; therefore, ~50 bp are present between the natural 5' end of exon 7 and the 3' splice site of the dsx gene. Since ESEs typically function in a distance-dependent manner (22,24,31), this additional distance may disrupt the regulatory elements within SMN exon 7. Secondly, the CT disruption may be subtle enough that in the absence of a downstream competitive splice site and exon (i.e. SMN exon 8), inclusion of exon 7 occurs essentially by default. Thirdly, the dsx cassette is designed to assess the relative strength of enhancers (22,24,31) and in a single copy neither SMN1 nor SMN2 is sufficient and the CT transition represents a disruption that was not measured in this context. Designing an in vitro template that recapitulates the in vivo SMN1 and SMN2 splicing patterns represents an important step in dissecting the biochemistry of exon 7 regulation.
The exon-skipped protein product is less stable and cannot provide protection from development of SMA
SMA develops as a consequence of the lack of a single functioning copy of SMN1, which expresses abundant full-length transcripts and no detectable levels of 7. Interestingly, even multiple copies of SMN2 that arise through partial recombination events cannot fully protect from SMA (39). The primary product of SMN2 is the exon-skipped product 7 RNA (1,9). We have reported that the 7 product is defective in self-association (37) and has been shown to be unable to stimulate in vitro splicing reactions like full-length SMN (7). This work identified a critical genetic regulatory mechanism for the production of 7 RNA. The protein product of the aberrant splicing event was much less stable than full-length SMN (Fig. 5). The stability of each protein form may be linked to their abilities to self-associate or to associate with additional cellular factors such as the Sm proteins (5,6,37). Potentially a more dramatic difference in protein stability would have been seen within a cellular background that lacked full-length endogenous SMN. SMN self-association is mediated by the peptide encoded by exon 6. Since the endogenous full-length SMN would be able to associate with the transiently expressed 7 product through the intact self-association domain encoded by exon 6 (37), the hetero-oligomers may form more stable complexes than 7 homo-oligomers. To date, all cell types examined contain full-length SMN, including type I SMA patients, even though endogenous SMN expression is typically decreased in cells derived from these types of patient (35,36). The relative instability of 7 may also contribute to disease development by binding to and subsequently destabilizing full-length SMN in a hetero-oligomer, thus explaining why even four copies of SMN2 fail to protect fully from SMA. Transiently expressed SMN7 protein has recently been detected 48 h post-transfection (40); however, this observation was based on steady-state protein levels, not cycloheximide treatment or pulse–chase analysis.
SMN1 and SMN2 are dramatically distinct functionally, yet at the nucleotide level they are nearly identical. Therefore, gaining insight into the mechanism(s) that regulate exon 7 inclusion versus exclusion is important in understanding SMA development. We have previously shown that the exon 7+6 nucleotide is critical for exon 7 inclusion, and now identify an essential ESE within exon 7. Interestingly, a very small number of SMA patients are not homozygously deleted for SMN1 (39), raising the possibility that SMA-modifying genes other than SMN2 exist (41). In further support of SMA-modifying genes (in addition to SMN2), SMA patients have been identified who contained the same mutated SMA allele; however, the clinical course of the disease was different. These results suggest that the factor(s) regulating exon 7 inclusion, including regulation through the AG-rich element, may be candidates for SMA-modifying genes. Identifying the factor(s) mediating SE2 responsiveness may lead to new insights into the pathogenesis of SMA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids
Previously described pSMN1 and pSMN2 mini-genes (8) were used as templates to generate oligo-mediated site-specific mutations with Thermo Pol Vent polymerase (New England Biolabs, Beverly, MA) using the splicing of overlapping extensions strategy, and cloned into the appropriate plasmid backbone between the BclI (SMN intron 6) and NotI (pCI polylinker) sites. The following primer sets were used to generate the indicated mutations (in bold):
pSMN1(C/A): 5'-CCT TAC AGG GTT TAA GAC AAA ATC-3';
5'-GAT TTT GTC TTA AAC CCT GTA AGG-3';
pSMN1(C/G): 5'-CCT TAC AGG GTT TGA GAC AAA ATC-3';
5'-GAT TTT GTC TCA AAC CCT GTA AGG-3';
SE1: 5'-CCT TAC AGG GTT TCA TTC TTA ATC AAA AAG AAG GAA GG-3';
5'-CCT TCC TTC TTT TTG ATT AAG AAT GAA ACC CTG TAA GG-3';
SE2: 5'-CAG ACA AAA TCA ATT ATT ATT AAG GTG CTA C-3';
5'-GTG AGC ACC TTA ATA ATA ATT GAT TTT GTC TG-3';
SE3: 5'-GGA AGG TGC TCT CTT TCC TTT TTT TAA GGA GTA AGT CTG CCA GC-3';
5'-GCT GGC AGA CTT AC TAC TCC TTA AAA AAA GGA AAG AGA GCA CCT TCC-3';
12Tppt: 5'-CTT CCT TTT TTT TTT TTA CAG GGT TTT/C AGA-3';
5'-TCT G/AAA ACC CTG TAA AAA AAA AAA AGG AAG-3';
SE2a: 5'-CAA AAT CAA TTT GAA GGA AGG TGC-3';
5'-GCA CCT TCC TTC AAA TTG ATT TTG-3';
SE2b: 5'-CAA AAT CAA AAA TTT GGA AGG TGC TCA C-3';
5'-GTG AGC ACC TTC CAA ATT TTT GAT TTT G-3';
SE2c: 5'-CAA AAA GAA TTT AGG TGC TCA CAT TCC-3';
5'-GGA ATG TGA GCA CCT AAA TTC TTT TTG-3'.
In vitro splicing cassettes were constructed by annealing the following pairs of complementary oligonucleotides that created XbaI and HindIII overhanging ends between the XbaI and HindIII sites of 3014, a plasmid that contains the previously described Drosophila double-sex splicing cassette (25):
dsx:SMN1 and SMN2: 5'-CTA GAG GTT TC/TA GAC AAA ATC AAA AAG AAG GAA GGT GCT CAC TT CCT TTA AAT TAA GGA A-3'; 5'-AGC TTT CCT TAA TTT AAG GAA TGT GAG CAC CTT CCT TCT TTT TGA TTT TGT CTG/A AAA CCT-3';
3xSE2: 5'-CTA GAC AAA AGA AGG AAG GCA AAA AGA AGG AAG GCA AAA AAG AAG GAA GGA A-3';
5'-AGC TTT CCT TCC TTC TTT TTGCC TTCCTT CTT TTT GCC TTC CTT CTT TTG T-3';
5xSE2: 5'-CTA GAA GAA GGA AGG AGA AGG AAG GAG AAG GAA GGA GAA GGA AGG AGA AGG AAG GAA-3';
5'-AGC TTT CCT TCC TTC TCC TTC CTT CTC CTT CCT TCT CCT TCC TTC TCC TTC CTT CTT-3'.
Mammalian expression vectors pCI:HA-SMN and -SMN7 were constructed by cloning the HA:SMN or 7 containing fragment from pcDNA3:HA:SMN or 7 (KpnI and a Klenow-filled XhoI fragment) into pCI (KpnI and SmaI).
Reverse transcription–PCR (RT–PCR)
U2OS cells, a human osteosarcoma line, were transiently transfected by standard CaPO4 procedures with 10 µg of the indicated plasmid. Thirty-six hours post-transfection, total RNA was isolated in guanidine thiocyanate lysis buffer and purified over a CsCl gradient. First-strand cDNA synthesis and amplification of plasmid-derived cDNAs were performed as previously described (8) except that pCI:Fwd#2 (5'-CGA CTC ACT ATA GCC TAG CC-3') was used in the PCR step, only 15 cycles were performed, and 40 µM dATP supplemented with 1 µl of [32P]dATP (3000 Ci/mmol; NEN, Boston, MA) were used. Reaction products were boiled and resolved in a 6% sequencing gel. Quantitations were performed on a BioRad (Hercules, CA) PhosphorImager; full-length and 7 transcripts were quantitated and expressed as a percent relative to full-length expression within the same reaction.
In vitro splicing
The Promega (Madison, WI) RNA Splicing System was used as recommended by the manufacturer. Briefly, in vitro splicing reactions were performed at 30°C for 3 h using 10 µl of HeLa nuclear extract and ~10 ng of gel-purified [32P]dUTP-labeled RNA templates. Reaction products were phenol extracted twice, precipitated and resolved on a 6% sequencing gel. Positive control reactions resulted in similarly sized splice products and DNA sequencing reactions were used to identify the final products.
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ACKNOWLEDGEMENTS |
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We thank Tom Maniatis and laboratory members, Mark Roth, Adrian Krainer and David Pintel for helpful discussions, and Carl Baker for the double-sex construct and advice. C.L.L. was supported by a fellowship from the Muscular Dystrophy Association. Funding for these studies was provided by the Muscular Dystrophy Association and Families of SMA to E.J.A.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 617 636 1493; Fax: +1 617 636 6190; Email: eandroph@opal.tufts.edu
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REFERENCES |
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S. R. Lim and K. J. Hertel Modulation of Survival Motor Neuron Pre-mRNA Splicing by Inhibition of Alternative 3' Splice Site Pairing J. Biol. Chem., November 21, 2001; 276(48): 45476 - 45483. [Abstract] [Full Text] [PDF]
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Y. Hofmann, C. L. Lorson, S. Stamm, E. J. Androphy, and B. Wirth Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2) PNAS, August 15, 2000; 97(17): 9618 - 9623. [Abstract] [Full Text] [PDF]
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