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1 Division of Structural Cellular Biology, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
2 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136, USA
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Abstract |
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Introduction |
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FTDP-17 is an autosomal-dominant disease with variable clinical and neuropathological features (Foster et al. 1997). Personality changes, sometimes with psychosis, hyperorality, reduced speech output and loss of executive function, are observed as symptoms (Lynch et al. 1994; Wilhelmsen et al. 1994; Wijker et al. 1996; Yamaoka et al. 1996; Spillantini et al. 1997; Bird et al. 1997; Heutink et al. 1997). Neuropathological changes include frontotemporal atrophy, sometimes with atrophy of the basal ganglion, substantia nigra and amygdala. FTDP-17 is caused by mutations in the tau gene, a microtubule-associated protein that normally functions to promote microtubule assembly and stability. In FTDP-17, tau protein aggregates in the brain to form abnormal filamentous structures including neurofibrillary tangles (Spillantini et al. 1996; Reed et al. 1997), neuropil threads, glial tangles and dense intracellular deposits (Spillantini et al. 1997). The type and location of tau pathology varies between FTDP-17 families.
Mutations of the tau gene cause FTDP-17 by, at least, two different mechanisms. First, the mutations impair function of tau protein. Tau with G272V, P301L, V337M, or R406W mutations exhibits reduced affinity and capacity for microtubule binding and a reduced ability to facilitate microtubule polymerization compared with wild-type (Hong et al. 1998). Second, the mutations cause aberrant splicing of tau exon 10. Some mutations in exon 10, or intron 10 immediately adjacent to the 3' end of the alternatively spliced exon 10, promote inclusion of exon 10 (Hutton et al. 1998; Spillantini et al. 1998; Hasegawa et al. 1999). Exon 10 encodes one of four microtubule-binding motifs found in the longer isoforms of tau protein. When exon 10 is included, an isoform with four microtubule-binding domains (4R tau) is produced, whereas exon 10 is excluded, an isoform with three microtubules repeats (3R tau) is produced. Alteration of the 3R/4R tau ratio impairs the normal tau function and induces on-set of FTDP-17, suggesting that the delicate balance between 3R and 4R tau isoforms is critical for neuronal function for maintaining learning and memory (Hutton et al. 1998; Hong et al. 1998; Goedert et al. 1998; Jiang et al. 2000; Goode et al. 2000).
To date, it has been reported that the regulatory elements that affect splicing of tau exon 10 exist at the 5' end of the exon and in the RNA stem-loop structure at the 3' end of exon 10. The 3' stem-loop structure modulates alternative splicing by restricting the accessibility of U1 snRNP to the downstream 5' splice site (Hutton et al. 1998; D’Souza et al. 1999; Grover et al. 1999; Jiang et al. 2000; Varani et al. 2000). Regulatory elements at the 5' end of exon 10 have been speculated to modulate the use of the weak 5' splice site of exon 10 (Clark et al. 1998; D’Souza et al. 1999; Hasegawa et al. 1999; Spillantini et al. 2000; Iseki et al. 2001). FTDP-17-related mutations are accumulated in this region, including N279K, 
280K and L284L, and are appeared to disrupt an exonic splicing enhancer (ESE) or an exonic splicing silencer (ESS). Some of ESE are the target sites of the specific serine/arginine-rich (SR) proteins (Graveley 2000). Recently, human Tra2ß, an SR-like protein, was demonstrated to interact with the 5' end of exon 10 and stimulates splicing (Jiang et al. 2003). However, it is unclear whether the sequences of the 5' end of exon 10 possess responsible ESE or whether other factors different from human Tra2ß stimulate splicing through the binding to the site. In this report, to examine the splicing mechanisms of tau exon 10, including both wild-type and disease-linked mutations, we investigated the function of multiple ESE motifs of the 5' side of exon 10 and its binding factors that regulate splicing of exon 10. We demonstrated that not only human Tra2ß but also SF2/ASF and SRp30c promoted the inclusion of exon 10 through association with the ESE at the 5' end of exon 10.
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Results |
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The mutations of tau gene used in this study are shown in Fig. 1A. These exon 10 fragments containing flanking intron sequences were subcloned between HIV tat exons and were transfected into neuroblastoma SK-N-SH cells. Twenty-four hours after transfection, total cellular RNA was isolated. Spliced products were detected by RT-PCR using the SD6 and SA2 primer set as described in the Experimental Procedures. When the wild-type exon 10 was tested, transcripts with both inclusion and exclusion of exon 10 were produced (Fig. 1B). The ratio of inclusion was 22% of the total and was similar level to that of the endogenous contents of tau products (Fig. 1C). Transfection of tau exon 10 containing FTDP-17-linked mutations, N279K and L284L, resulted in an increase of the inclusion to 78% and 69% of the total, respectively (Fig. 1B). In contrast, transfection with the 280K mutation led to no detectable inclusion of exon 10. These results suggest that mutations linked to FTDP-17 significantly affect splicing of tau exon 10. We also examined the splicing pattern of tau exon 10 in non-neuronal cells such as HEK293T or COS-7. The results were basically the same as those in the neuronal cell lines (data not shown), indicating that the splicing of tau exon 10 is regulated by the same mechanism in both neuronal and non-neuronal cells.
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The cis-element of the exon, which stimulates splicing, was designated as an exonic splicing enhancer (ESE) and often contains an AG-rich sequence (Blencowe 2000; Cartegni et al. 2002). The putative AG-rich ESE sequence (wild-type tau, AATAAGAAG) is located at the 5' side of tau exon 10. Mutations linked to FTDP-17 are accumulated in this region or are close to the sequence. We examined the mechanism of tau exon 10 splicing containing FTDP-17-linked mutations in the putative ESE sequence. To examine whether the 5' side of exon 10 had ESE function, we joined a 34-nt sequence including the 5' side of tau exon 10 (nucleotides 12-45) into the downstream exon of an enhancer-dependent splicing reporter substrate derived from the D. melanogaster dsx gene (Fig. 2A). The intron in the dsx system is efficiently spliced only when an SR-dependent ESE is present in the downstream exon (Tian & Maniatis 1992, 1994; Tanaka et al. 1994; Graveley et al. 1998). Splicing of the dsx substrate (dsx-E) was extremely weak (Fig. 2B). As a control, a strong SR-dependent splicing enhancer derived from the avian sarcoma-leukosis virus (ASLV) was inserted into the downstream exon of the dsx substrate (dsx-ASLV) and this pre-mRNA was spliced efficiently (Fig. 2B). Insertion of the wild-type 5' region of exon 10 into the dsx splicing reporter substrate caused weak splicing, suggesting that the 5' region of tau exon 10 functioned as the weak ESE. Insertion of mutants (N279K or L284L) induced marked increase of splicing to an equivalent level of that obtained with the ASLV enhancer. In contrast, the 280K mutant completely disrupted the ESE function (Fig. 2B). The splicing pattern in each mutant were very similar to those of the exon trapping system (Fig. 1B), indicating that mutations such as N279K, L284L and 280K affect the ESE function and change the splicing pattern of tau exon 10.
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The mutations linked to FTDP-17 are accumulated at the 5' side of tau exon 10, which includes active ESE as shown in Fig. 2B. Some of ESE sequences are the target sites of SR-related proteins, therefore, SR-related proteins could regulate incorporation of exon 10 through direct binding to the ESE. To identify the responsible SR proteins that regulate splicing of exon 10, we examined the effects of various SR proteins on the splicing of exon 10 in vivo using an exon trapping system. Both the exon trap vector and expression plasmids for various SR proteins were co-transfected into HEK293T cells; 30 h after transfection, total RNA was collected and subjected to RT-PCR. Among the SR proteins examined, the expression of human Tra2ß, SF2/ASF and SRp30c promoted inclusion of tau exon 10 compared with the mock-transfected cells (Fig. 3). In contrast, the other SR proteins did not affect the splicing, suggesting that these three SR proteins could be candidates for the ESE-dependent activators of exon 10-inclusion. Furthermore, we confirmed that these proteins are expressed in mouse brains by RT-PCR (data not shown).
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280K mutant substrate.
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We have demonstrated that the 5' side of tau exon 10 includes active ESE and SR proteins, human Tra2ß, SF2/ASF and SRp30c, promoted incorporation of exon 10. Therefore, we next examined whether these SR proteins really associated with exon 10. To this end, various 32P-labelled RNA substrates including exon 10 sequences were incubated with nuclear extracts that were prepared from HEK293T cells transfected with expression plasmids of FLAG-tagged SR proteins. The complexes were immunoprecipitated with an anti-Flag monoclonal antibody, washed extensively and bound fractions were analysed by scintillation counting. We confirmed that transfection efficiency or expression levels of each tagged-SR protein were almost equivalent by Western blotting (Fig. 5B). We could detect that tau exon 10 fragment was significantly associated with human Tra2ß, SF2/ASF and SRp30c compared with the background level of binding (Fig. 5A). The amount of each SR protein bound to the tau RNA were in order as SRp30c, SF2/ASF and human Tra2ß. This observation is consistent with the previous report that showed human Tra2ß interacts with the AG-rich region of tau exon 10 (Jiang et al. 2003). However, the amounts of Tra2ß interacting with RNA were at comparably lower levels, in contrast, it is of interest that the amounts of bound SF2/ASF and SRp30c were at rather higher levels.
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280K or L284L mutations did not alter the amount of binding of any of the SR proteins (Fig. 5C), indicating that the changes in ESE function caused by the L284L and 280K mutations are not dependent on these SR proteins, but may be dependent on other factors. |
Discussion |
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280K, have been reported to alter the ESE function and result in changes in the normal splicing pattern of tau exon 10 (D’Souza et al. 1999; Clark et al. 1998). However, the detailed mechanism of this aberrant splicing has not been elucidated. Recently, human Tra2ß, one of the SR-like proteins, was demonstrated to interact with the 5' side of tau exon 10 and to stimulate its splicing (Jiang et al. 2003). In this report, utilization of the suboptimal 3' splice site was only detectable in splicing assay in vitro when additional U2AF65 protein with human Tra2ß was provided in the splicing reactions. Therefore, it is unclear whether human Tra2ß really plays a role in stimulating splicing of exon 10 through binding to the ESE sequence. Here we successfully demonstrated that recognition of the weak 3' splice site of exon 10 was promoted by the ESE, that was located in nucleotides 12-45 of exon 10 by in vitro splicing assay. Furthermore, FTDP-17-linked mutations such as N279K, 280K and L284L were shown to affect the function of this ESE and result in aberrant splicing of exon 10.
The 5' site of exon 10 as the ESE contains an AG-rich sequence, AATAAGAAG. The sequence in the N279K mutation, which is linked to FTDP-17, is AAGAAGAAG. In the binding assay, this mutation resulted in increased amounts of human Tra2ß bound to tau exon 10 compared with the wild-type. Based on previous evidence, it is conceivable that the binding activity of Tra2ß to the ESE may correlate with the copy number of GAA repeats in the ESE (Tacke et al. 1998). In contrast, the human Tra2ß binding to the 280K mutant pre-mRNA (AATAAG) was not altered compared with that of the wild-type. Nevertheless, the ESE function was completely disrupted in 280K mutant, suggesting that the skipping of exon 10 by the 280K mutation may be caused by human Tra2ß-independent mechanism. It is possible that the binding of other SR proteins to the ESE could be inhibited by the 280K mutation, or a splicing repressor may turn to bind to 280K mutant pre-mRNA.
SF2/ASF was also associated well with tau pre-mRNA containing N279K compared with the wild-type sequence, but there were no significant changes in SF2/ASF binding between the wild-type and 280K. The high score of the ESE motif analysis program (threshold values were: SF2/ASF heptamer motif, 1.956) showed that the ESE sequence of wild-type or N279K mutant does not contain any sequences that are over the threshold value of SF2/ASF. These findings suggest that SF2/ASF is not directly associated with tau pre-mRNA through the ESE, and rather that the increase of SF2/ASF binding to tau pre-mRNA may be mediated by other RNA binding proteins. The in vitro splicing assay showed that SF2/ASF itself stimulated splicing of the wild-type tau exon 10, similar to human Tra2ß. Therefore, it is likely that human Tra2ß functionally cooperates with SF2/ASF and indir ectly binds to the AG-rich ESE in exon 10 to promote inclusion of the exon. However, further investigation is necessary to determine the detailed mechanism responsible for the splicing regulation of tau exon 10 by SF2/ASF associated with human Tra2ß.
SRp30c is a member of the authentic SR protein family although its mechanism of action has not been documented (Screaton et al. 1995; Stoss et al. 1999; Estmer Nilsson et al. 2001). In this study, over-expression of SRp30c in HEK293T cells induced inclusion of exon 10 in vivo and it also promoted splicing of the dsx-tau heterologous substrate. These findings indicate that SRp30c may be involved in the regulation of the ESE function of tau exon 10 splicing. However, the amount of SRp30c bound to the wild-type pre-mRNA was not altered compared with those of various mutations (N279K, 280K, L284L). Therefore, SRp30c is presumably associated indirectly with the tau pre-mRNA. Previously, SRp30c was reported to associate with SMN exon 7 in a trimeric complex through a direct interaction with human Tra2ß (Young et al. 2002). As shown in our study and another report (Jiang et al. 2003), human Tra2ß directly binds to the ESE sequence of tau exon 10 pre-mRNA and promotes splicing of tau exon 10, indicating that SRp30c may associate with the ESE sequence through human Tra2ß. It is possible that SRp30c may stabilize interaction between human Tra2ß and ESE through protein-protein interaction and it may help to define exon 10 to be included. We further examined the interaction of SF2/ASF and SRp30c on the pre-mRNA of tau exon 10, but could not successfully demonstrate its interaction. Further examinations are needed to understand the detailed mechanisms of splicing regulation by the association of SF2/ASF and SRp30c.
The L284L mutation also activated the ESE and increased the inclusion of tau exon 10 as well as N279K mutant. The amount of both Tra2ß and SF2/ASF binding to exon 10 with N279K mutation was increased compared with those of wild-type, but the amount of binding to L284L mutant RNA were not changed. This suggests that other RNA binding proteins, besides human Tra2ß and SF2/ASF could bind to tau exon 10 with L284L mutation and activate ESE by different mechanisms. The ESE motif analysis showed that the L284L mutation resulted in a much higher score of SRp40, that was over the threshold score (wild-type, TCTTAGC, score = 2.05; L284L, TCTCAGC, score = 4.43). The score alteration indicates the possibility that the mutation leads to binding of SRp40 to tau exon 10 including L284L mutation and the SRp40 binding may be responsible for the inclusion of exon 10.
In summary, we determined the functional ESE in the 5' side of tau exon 10 (nucleotides 12-45) by in vitro splicing with the heterologous dsx-substrate. We demonstrated that the FTDP-17-linked mutations indeed affect the ESE function and change the splicing pattern of exon 10. Human Tra2ß binds directly to the ESE sequence including the N279K mutation and stimulates inclusion of exon 10. SF2/ASF also binds to wild-type tau exon 10, its binding is increased by N279K mutation and eventually promotes the splicing, indicating that SF2/ASF cooperates functionally with human Tra2ß which binds to the AG-rich ESE in the N279K mutant. SRp30c binds to wild-type tau exon 10 and strongly stimulate splicing of exon 10, but the detailed mechanism remains to be elucidated. We have studied a mechanism of an ESE motif in tau exon 10 and how exon inclusion is induced by FTDP-17-linked mutations. Though further investigation is required, it may be possible to modulate this mutated ESE function by using stable decoy RNA, harbouring these mutations, which may sequester the proteins that alter this ESE function and may lead to a therapeutic strategy for FTDP-17.
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Experimental procedures |
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COS-7, HEK293T and SK-N-SH cells were used for the in vivo splicing assays. COS-7 and HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and SK-N-SH cells were cultured in Alpha Minimum Essential Medium (
-MEM) with 10% FCS. Prior to transfection, cells were plated at a density of 60% to 80% confluency on 3.5 cm dishes.
Exon trapping systems
Tau mini-genes (both wild-type and mutants) containing intron 9, exon 10 and intron 10 were subcloned into the Exon Trapping Vector pSPL3 (Life Technologies) (Church et al. 1994; Miyajima et al. 2002) or a modified exon trapping vector driven by a CMV promoter. All constructs were verified their sequence before use in the experiments. The plasmids were transfected into SK-N-SH cells and total cellular RNA was isolated 24 h after transfection using an RNeasy Mini Kit (Qiagen). Aliquots of 3.0 µg RNA were reverse-transcribed using the SA2 primer (5'-ATC TCA GTG GTA TTT GTG AGC-3') and MMLV reverse transcriptase (Life Technologies). Spliced products were detected by PCR using pSPL3 vector-specific primer sets, SD6 (5'-TCT GAG TCA CCT GGA CAA CC-3') and SA2. PCR (in 40 µL) was performed as follows: 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C for 30 cycles, followed by 72 °C for 5 min. The PCR products were analysed by 5% polyacrylamide gel electrophoresis (PAGE).
The cDNAs of SR proteins were amplified from human SK-N-SH cells, tagged with Flag-epitope at the C-terminus and cloned into pCDNA 3.1 expression vectors (Invitrogen). Flag-tagged-SC35 and -9G8 were cloned into pCDNA 3.1 constructed from the templates inserted into pCGT7 (a gift from Dr Krainer, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, USA).
For analysis of splicing of endogenous tau in SK-N-SH cells, RT-PCR was performed using a primer set (forward: 5'-AAGGTGGCAGTGGTCCGTACTC-3'; reverse: 5'-GACCCAATCTTCGACTGGACTC-3').
RNA-protein binding assays
Quantitative binding assays were performed as previously described (Young et al. 2002). Briefly, sense strand RNA corresponding to tau exon 10 was transcribed in vitro with [-32P] UTPs. RNAs were incubated at 4 °C overnight with cellular extracts that were prepared from HEK293T cells (in 10 cm dishes) transfected with expression plasmids (10 µg) of Flag-tagged SR proteins and then were immunoprecipitated by anti-FLAG monoclonal antibody. Bound fractions were determined using a scintillation counter.
In vitro splicing
A 34-nt fragment (nucleotides 12-45) of the 5' side of tau exon 10 were subcloned into the downstream exon 4 of an enhancer-dependent splicing reporter substrate, which was derived from the D. melanogaster dsx gene (a gift from Dr K. Inoue, Kobe University, Kobe, Japan) (Tanaka et al. 1994). Capped and 32P-labelled run-off transcripts were synthesized by in vitro transcription using T7 RNA polymerase (Promega). Splicing reactions (in 25 µL) containing 10 µL of HeLa cell nuclear extracts (Promega) were performed as previously described (Mayeda & Krainer 1999). The splicing mixtures were incubated at 30 °C for the indicated time. To analyse spliced products, RNA recovered from the splicing reaction mixtures was reverse-transcribed by primer sets (dsx-E, 5'-AGGATCCCCGACGGGAG-3'; dsx-ASLV, 5'-TCCTTCTTGCTTGTTGCTGGCG-3'; dsx-tau WT, N279K, 280K, 5'-AAGCTTGGACTGGACGTTGC-3'; dsx-tau L284L, 5'-AAGCTTGGACTGGACGTTGC-3') and following PCR was performed using primer sets (dsx-E, forward: 5'-GCCAAGACGTTTTCCTAG-3', reverse: 5'-AGGATCCCCGACGGGAGTACTC-3'; dsx-ASLV, forward: 5'-GCCAAGACGTTTTCCTAG-3', reverse: 5'-TCCTTCTTGCTTGTTGCTGGCGGCTT-3'; dsx-tau WT, N279K and 280K forward: 5'-GCCAAGACGTTTTCCTAG-3', reverse: 5'-GGACTGGACGTTGCTAAGATCCAGC-3'; dsx-tau L284L, forward: 5'-GCCAAGACGTTTTCCTAG-3', reverse: 5'-GGACTGGACGTTGCTGAGATCCAGC-3'). Recombinant SR proteins tagged with glutathione S-transferase (GST) were prepared from E. coli and added to the splicing reactions.
High-score motif analysis
We analysed the exon sequences from tau and its mutants as previously described (Liu et al. 2001; Cartegni & Krainer 2002). For each SR protein, we calculated the highest score for each sequence in a pool of 30 random 20-mers and set the median of these high scores as the threshold value. The threshold values were: SF2/ASF heptamer motif, 1.956; SRp40 heptamer motif, 2.670; SRp55 hexamer motif, 2.676; SC35 octamer motif, 2.383.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: imaizumi{at}bs.aist-nara.ac.jp
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References |
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Received: 5 September 2003
Accepted: 8 December 2003
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200 ng). The scores shown in the lower panel are the percentage of the spliced products relative to the total transcripts.