SIRT1 Deacetylates SC35 and Suppresses Its Function in Tau Exon 10 Inclusion

Abstract

Approximately equal amounts of 3R-tau and 4R-tau resulting from alternative splicing of tau exon 10 is necessary to maintain normal brain function. Dysregulation of alternative splicing of tau exon 10 and the imbalance of 3R-tau/4R-tau have been seen in inherited and sporadic tauopathies. Splicing factor SC35 (also name as SRSF2) plays an important role in promoting tau exon 10 inclusion. SC35 is post-translationally modified by phosphorylation and acetylation, but the role of acetylation in SC35-medicated tau exon 10 inclusion is unknown. Sirtuin type 1 (SIRT1) is an enzyme that deacetylates proteins and associates with age-related disease such as Alzheimer’s disease. In the present study, we determined the role of SIRT1 in SC35 acetylation and in the alternative splicing of tau exon 10. We found that SIRT1 interacts with and deacetylates SC35, and inhibits SC35-promoted tau exon 10 inclusion. Substituting K52 residue of SC35 by arginine impairs the role of SC35 in tau exon 10 inclusion. These results suggest that SIRT1 may serve as a therapeutic target for tauopathy by regulating SC35-mediated tau exon 10 splicing.

Keywords

alternative splicing deacetylation SC35 SIRT1 tau exon 10

INTRODUCTION

Tau protein is the major microtubule-associated protein in the central nervous system and plays a critical role in the assembly and stability of microtubules. Hyperphosphorylated tau aggregates to form paired helical filaments, which is the major component of neurofibrillary tangles in the brain of individuals with Alzheimer’s disease (AD) and associated tauopathies [1, 2]. Mutations in tau gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), indicating the pathologic changes of tau could trigger neurodegeneration [3, 4]. Adult human brain expresses six isoforms of tau due to the alternative splicing of tau exons 2, 3, and 10 from the single gene. Tau exon 10 encodes the second microtubule binding repeat of tau protein. Its exclusion or inclusion generates tau isoforms with three or four microtubule binding repeats, termed as 3R-tau or 4R-tau [5, 6]. In normal human brain, the ratio of 3R-tau/4R-tau is about 1. However, in some brains of individuals with FTDP-17, tau exon 10 is abnormally alternative spliced due to certain mutations of tau gene, resulting in the imbalance of 3R-tau/4R-tau and consequently, the hyperphosphorylation and aggregation of tau and neurofibrillary degeneration [7]. The imbalance of 3R-tau/4R-tau is also observed in other tauopathies, such as Down syndrome, corticobasal degeneration, Pick’s disease, and progressive supranuclear palsy [7, 8]. Therefore, approximately equal amount of 3R-tau and 4R-tau is important for maintaining normal brain function.

Previous reports indicate that multiple splicing factors participate in the regulation of tau exon 10 splicing [9]. SC35 (also named as SRSF2) plays an important role in promoting tau exon 10 inclusion by acting on SC35-like exonic splicing enhancers on tau exon 10 [10, 11]. SC35 is a member of serine and arginine rich proteins (SR proteins) [12]. The functions and subcellular localization of SC35 are highly regulated by the phosphorylation [11 , 13– 15]. In addition to phosphorylation, it has been reported that SC35 is also acetylated by Tip60 at Lys 52. The acetylation affects its degradation and function in the regulation of caspase 8 alternative splicing [16].

Sirtuins are a kind of highly conserved deacetylases, which are closely relevant to aging and resistant to neurodegenerative disorders related to aging [17]. There are seven members in mammalian Sirtuin family, from SIRT1 to 7 [18]. They belong to class III histone deacetylases, the activity of which depends on the relative level of NAD⁺ and NADH [18, 19]. SIRT1 has been extensively studied and is well known for associating closely with age-related diseases such as AD [20]. The decrease of SIRT1 level in the AD brain is in parallel with the accumulation of tau [21]. SIRT1 not only reduces amyloid plaques but also suppresses symptoms related to tau effectively [22 –24]. Activation of SIRT1 may become the new strategy for preventing neurodegenerative disorders [19]. However, whether and how SIRT1 regulates SC35 acetylation and its function in the alternative splicing of tau exon 10 is unknown. In the present study, we investigated the role of SIRT1 in SC35 acetylation as well as its function in the regulation of tau exon 10 splicing. We found that SIRT1 deacetylated SC35 and suppressed SC35-promoted tau exon 10 inclusion.

METHODS

Plasmids and antibodies

pCEP4/SC35-HA was a gift from Dr. Tarn of the Institute of Biomedical Sciences, Academia Sinica, Taiwan. pCI/SI9-SI10 containing a tau minigene, SI9/SI10, comprising tau exons 9, 10, and 11, part of intron 9 and intron 10 was as described [25]. Mouse monoclonal anti-SIRT1 and Anti-acetylated-lysine were from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti-HA, mouse monoclonal anti-HA, and mouse monoclonal anti-actin were from Sigma (St. Louis, MO, USA). Mouse monoclonal anti-Myc, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, and human siRNA of SIRT1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-phosphoserine was from Invitrogen (Carsbad, CA, USA). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The ECL kit was from Thermo Scientific (Rockford, IL, USA).

Plasmid construction and DNA mutagenesis

pcDNA3.1-SIRT1 was constructed by subcloning SIRT1 coding region which was PCR amplified from Flag-SIRT1 plasmid purchased from Addgene (Cambridge, MA, USA) into mammalian expression vector pcDNA3.1 by BamHI and NotI. pGEX-2T/SC35 was constructed by PCR amplification from pCEP4/SC35 and subcloned into pGEX-2T by BamHI and XhoI to express GST-SC35 protein. The deletion mutations of SC35 were generated by amplifying an individual fragment, which contains part of the SC35 coding region into the HindIII-XhoI sites of pCEP4. SC35_K52R mutant was created by site-directed mutagenesis using KOD-Plus-Mutagenesis kit (TOYOBO, Osaka, Japan) with primers (forward, 5’- CCG CTA CAC CAG GGA GTC CCG CGG CTT CGC CTT CGT TC-3’, and reverse, 5’- CGC GGG ACT CCC TGG TGT AGC GGT CCC GCG GGA TGT AC-3’).

Cell culture and transfection

HEK-293FT and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen) at 37°C (5% CO₂). Transfections were performed with Lipofectamine 3000 (Invitrogen) or FuGene 6 (Roche, IN, USA), according to the manufacturer’s instructions.

GST pull down

GST or GST-SC35_FL, GST-SC35_1 - 191, GST-SC35_1 - 117, GST-SC35_88 - 221 was purified by affinity purification with glutathione-Sepharose without elution from the beads. Beads coupled with GST or GST-SC35_FL, GST-SC35_1 - 191, GST-SC35_1 - 117, GST-SC35_88 - 221 were incubated with crude extract from rat brain homogenate in buffer (50 mM Tris-HCl, pH 7.4, 8.5% sucrose, 50 mM NaF, 1 mM Na₃VO₄, 0.1% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10μg/ml aprotinin, 10μg/ml leupeptin, and 10μg/ml pepstatin). After a 4 h incubation at 4°C, the beads were washed with washing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM dithiothreitol) six times and the bound proteins were eluted by boiling in Laemmli sample buffer and analyzed by western blot analysis.

Co-immunoprecipitation

HEK-293FT cells were co-transfected with pcDNA3.1/SIRT1 and pCEP4/SC35_FL-HA, pCEP4/SC35_1 - 191-HA, pCEP4/SC35_1 - 117-HA or pCEP4/SC35_88 - 221-HA for 48 h. The cells were washed twice with phosphate-buffered saline (PBS) and lysed by sonication in lysate buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2μg/ml aprotinin, 2μg/ml leupeptin, and 2μg/ml pepstatin). Insoluble materials were removed by centrifugation. Protein G beads were incubated with anti-HA overnight at 4°C, and then the antibody bound beads were incubated with the cell lysate. After a 4 h incubation at 4°C, the beads were washed with lysate buffer twice and with Tris-buffered saline twice, and bound proteins were eluted by boiling in Laemmli sample buffer. The samples were subjected to western blot analysis with the indicated primary antibodies.

Co-localization study

HeLa cells were plated in 24-well-plates onto coverslips 1 day prior to transfection at 30– 40% confluence. These cells were transfected with HA-tagged SC35 constructs or co-transfected with SIRT1 as described above. Two days after transfection, the cells were washed with PBS and fixed with 4% paraformaldehyde in PB for 30 min at room temperature. After washing with PBS, the cells were blocked with 10% goat serum in 0.2% Triton X-100/ PBS for 2 h at 37°C and incubated with rabbit anti-HA (1:200) and mouse anti-Myc (1:500) overnight at 4°C. After washing with PBS and incubation with secondary antibodies (TRITC-conjugated goatanti-rabbit IgG and FITC-conjugated goat anti-mouse IgG, 1:200), the cells were washed extensively with PBS and incubated with 5μg/ml Hoechst 33342 for 5 min at room temperature. The cells were washed with PBS, mounted with Fluoromount-G, and visualized with a Leica TCS-SP2 laser-scanning confocalmicroscope.

Quantitation of tau exon 10 splicing by reverse transcription-PCR (RT-PCR)

Total cellular RNA was isolated from cultured cells by using an RNeasy mini kit (Qiagen, GmbH,Germany). Six hundred ng of total RNA was used for first-strand cDNA synthesis with oligo (dT)18 by using an Omniscript reverse transcription kit (Qiagen). PCR was performed by using Prime-STARTM HS DNA Polymerase (Takara Bio Inc., Otsu, Shiga, Japan) with primers (forward 5’ GGT GTC CAC TCC CAG TTC AA 3’ and reverse 5’ CCC TGG TTT ATG ATG GAT GTT GCC TAA TGA G 3’) to measure alternative splicing of tau E10 of mini-tau gene under conditions: denaturation for 5 min at 98°C was followed by 30 cycles with denaturation for 10 s at 98°C, annealing for 15 s at 55°C, polymerization for 30 s at 72°C, and a final extension for 10 min at 72°C. The PCR products were resolved on 1.5% agarose gels and quantitated using the Molecular Imager system (Bio-Rad, CA, USA).

Statistical analysis

Where appropriate, the data are presented as the means±S.E.M. Data points were compared by the unpaired two-tailed Student’s t-test for two groups’ comparison and one way ANOVA for multiple groups’ comparison. The calculated p-values are indicated in the figures.

RESULTS

Acetylation status of SC35 modulates the alternative splicing of tau exon 10

SC35 regulates the alternative splicing of tau exon 10 [26]. To confirm the role of SC35 in tau exon 10 splicing, we co-transfected mini-tau gene pCI/SI9-SI10, consisting of tau exon 9, 10, and 11, part of intron 9 (SI9) and intron 10 (SI10) with SC35, ASF, SRp55, SRp30c, or SRp40 expression plasmids into HEK-293FT cells. Total RNA was extracted from the cells and the alternative splicing products of tau exon 10 were examined by RT-PCR 48 h after transfection. We found that SC35 had much more significant influence on tau exon 10 splicing compared with the rest (Fig. 1A), supporting the important role of SC35 in tau exon 10 inclusion.

Fig.1

Acetylation influences SC35’s function in promotion of tau exon 10 inclusion. A) Different kinds of SR proteins were co-transfected with pCI/SI9-SI10 into HEK-293FT cells. After 48 h transfection, total RNA was extracted and the alternative splicing products of tau exon 10 were examined by RT-PCR. Expression levels of SR proteins were analyzed by western blots developed with anti-HA. GAPDH was used as a loading control. B) SC35 or SC35_K52R tagged with HA was expressed in HEK-293FT cells, immunoprecipitated with anti-HA, and analyzed by western blots developed with anti-HA or anti-acetylated-lysine. C) SC35_K52R was deficient in promoting tau exon 10 inclusion. pCEP4/SC35 or pCEP4/SC35_K52R was co-transfected with pCI/SI9-SI10 into HEK-293FT cells. After 48 h transfection, total RNA was extracted. The alternative splicing products of tau exon 10 were analyzed by RT-PCR. The ratio of inclusion and exclusion of tau exon 10 was calculated and are represented as mean±S.E.M. (n = 3); ^***p < 0.001.

It was reported that SC35 is acetylated by the acetyltransferase Tip60 on its lysine 52 [16]. To determine the acetylation of SC35 or SC35_K52R, we mutated lysine 52 of SC35 into arginine, and overexpressed HA-SC35 or HA-SC35_K52R in HEK-293FT cells. We immunoprecipitated them with anti-HA antibody and analyzed the acetylation status of SC35 or SC35_K52R with western blots after 72 htransfection. We found that both SC35 and SC35_K52R were acetylated (Fig. 1B). However, compared with SC35, the acetylation level of SC35_K52R was obviously decreased (Fig. 1B). Thus, these results indicate that SC35 is acetylated on, but not only on Lys52.

To learn the role of SC35 acetylation on K52, we co-transfected pCEP4/SC35 or pCEP4/SC35_K52R together with pCI/SI9-SI10 into HEK-293FT cells. We analyzed the alternative splicing products of tau exon 10 by RT-PCR 48 h after transfection. We found that both SC35 and SC35_K52R enhanced tau exon 10 inclusion, though the role of SC35_K52R is weaker than that of SC35 (Fig. 1C).

SIRT1 interacts with SC35

SIRT1 deacetylates many proteins and associates closely with age-related diseases. To know whether SIRT1 interacts with SC35, we overexpressed SIRT1 with Myc-tag and SC35 with HA-tag in HEK-293FT cells and immunoprecipitated SIRT1 with anti-Myc. Then, we analyzed whether SC35 was co-immunoprecipitated by SIRT1 using western blots. We detected that SC35-HA was precipitated with anti-Myc in the immunocomplex (Fig. 2A), suggesting SC35 was co-immunoprecipitated with SIRT1.

Fig.2

SIRT1 interacts with SC35. A) Myc-tagged SIRT1 and HA-tagged SC35 were co-expressed in HEK-293FT cells. SIRT1 was immunoprecipitated by anti-Myc. The immunoprecipitated proteins were analyzed by western blots developed with anti-Myc or anti-HA. B) Myc-tagged SIRT1 and HA-tagged SC35 were co-overexpressed in HeLa cells. After 48 h transfection, the cells were immunostained by anti-HA or anti-Myc and followed by TRITC-anti-rabbit IgG or FITC-anti-mouse IgG. Hoechst was used for nuclear staining.

Both SIRT1 and SC35 are mainly expressed in nucleus [27, 28]. Thus, we used HeLa cells to study their subcellular localization. We co-expressed SIRT1-Myc and SC35-HA in HeLa cells and carried out immunofluorescence staining 48 h after transfection. We found that in the single plasmid transfected cells, either SC35 or SIRT1 mainly expressed in nuclei evenly (Fig. 2B). However, co-expression of SC35 promoted SIRT1 to translocate into periphery of the nucleus (Fig. 2B) and co-expression of SIRT1 made SC35 translocate to periphery of nuclei partially and co-localize with SIRT1 (Fig. 2B). These results suggest that SIRT1 may interact with SC35.

SIRT1 deacetylates SC35

To investigate the role of SIRT1 in SC35 acetylation, we co-transfected pCEP4/SC35 with pcDNA3.1/SIRT1 or siRNA of SIRT1, and immunoprecipitated SC35 with anti-HA antibody. The acetylation status of immunoprecipitated SC35 was analyzed by western blots using anti-acetylated-lysine antibody. As shown in Fig. 3A and B, the level of acetylated SC35 was significantly decreased in the cells with co-expression of SIRT1 and significantly increased in the cells with knockdown of SIRT1 by siSIRT1. Thus, these results indicate that SIRT1 deacetylates SC35.

Fig.3

SIRT1 deacetylates SC35. pCI/SC35 was co-transfected with pcDNA3.1/SIRT1 or siRNA of SIRT1 into HEK-293FT cells. SC35 was immunoprecipitated with anti-HA and analyzed by western blots developed with anti-HA or anti-acetylated-lysine (A) and anti-phospho-serine (C) antibody. The levels of SC35 acetylation (B) or phosphorylation (D) are represented as mean±S.E.M. (n = 3); ^**p < 0.01, ^***p < 0.001.

The function of SR protein is tightly regulated by the phosphorylation. SC35 contains many serine residues [12]. To learn whether SIRT1 influences the phosphorylation of SC35, we determined the phosphorylation level of immunoprecipitated SC35 with western blots developed by anti-phospho-serine. We found that either overexpression or knockdown of SIRT1 had no significant effect on the phosphorylation of SC35 (Fig. 3C, D), indicating that SC35 acetylation may not influence its phosphorylation.

Either RRM or RS domain of SC35 interacts with SIRT1

SC35 contains a N-terminal RNA recognition motif (RRM) and a C-terminal arginine/serine rich (RS) domain (Fig. 4A). To determine by which domain SC35 interacts with SIRT1, we made various deletion mutants of SC35 and expressed them as GST-fusion proteins in E. coli cells. The GST-fusion proteins were affinity-purified with Glutathione conjugated agarose beads without elusion and incubated with rat brain extract. The co-pull-down proteins by the GST-fusion proteins were analyzed by western blots developed with anti-SIRT1. We detected SIRT1 in the pull-down fractions by all GST-fusion SC35 and its deletion mutants, but not GST (Fig. 4B), suggesting SIRT1 may interact with SC35 by both RRM and RS domains of SC35. GST- SC35_1 - 191 without nuclear retention signal (NRS) domain pulled down more SIRT1 than other mutants (Fig. 4B), suggesting that C-terminal NRS domain may inhibit SC35 to interact with SIRT1 in vitro.

Fig.4

SIRT1 interacts with SC35 through RRM or RS domain. A) Schematic of SC35 deletion mutants. B) GST-SC35_FL, GST-SC35_1 - 191, GST-SC35_1 - 171, or GST-SC35_88 - 221 or GST coupled onto glutathione sepharose was incubated with rat brain extract. Pull-down proteins were analyzed by western blots developed with anti-GST and anti-SIRT1. C) Myc-SIRT1 was co-expressed with HA-SC35_FL, HA-SC35_1 - 191, HA-SC35_1 - 171, or HA-SC35_88 - 221 in HEK-293FT cells. SC35 and its mutants were immunoprecipitated by anti-HA. Co-immunoprecipitated proteins were analyzed by western blots developed with antibodies indicated at the right of each blot. SIRT1 in cell lysate was used as loading control (lower panel). D) Co-localization of truncation forms of SC35 with SIRT1 in nucleus. HA-SC35_FL, HA-SC35_1 - 191, HA-SC35_1 - 171, or HA-SC35_88 - 221 was co-transfected with Myc-SIRT1 respectively into HeLa cells. After 48 h transfection, the cells were immunostained by anti-HA or anti-Myc and followed by TRITC-anti-rabbit IgG or FITC-anti-mouse IgG. Hoechst was used for the staining of nuclei.

We then co-expressed HA-tagged SC35 and its deletion mutants with SIRT1 in HEK-293FT cells and performed co-immunoprecipitation assays with anti-HA. We found that SIRT1 was co-immunoprecipitated by SC35 and all its deletion mutants (Fig. 4C). More abundant SIRT1 was co-immunoprecipitated by SC35_1 - 191, in which C-terminal NRS was deleted (Fig. 4C). These results support that both RRM and RS domains interact with SIRT1. C-terminal NRS domain may interfere the interaction of SC35 with SIRT1.

To further study the interaction of SC35 with SIRT1 in intact cells, we overexpressed SC35 and its deletion mutants tagged with HA together with SIRT1 in HeLa cells and immunostained them. We found that SC35 and its deletion mutants, SC35_1 - 191, SC35_1 - 117, and SC35_88 - 221 was co-localized with SIRT1 at the peripheral nucleus (Fig. 4D), which further confirming both RRM and RS domains interact with SIRT1.

SIRT1 suppresses SC35-promoted tau exon 10 inclusion

SC35 enhanced tau exon 10 inclusion significantly (Fig. 1A). In order to determine whether SIRT1 affects SC35’s function in the regulation of tau exon 10 splicing, we co-expressed SIRT1 together with SC35 in HEK-293FT cells expressing pCI/SI9-SI10 mini-tau gene and analyzed tau exon 10 splicing. We found that overexpression of SC35 or SIRT1 enhanced tau exon 10 inclusion (Fig. 5A, B). However, co-expression of SIRT1 suppressed SC35-enhanced tau exon 10 inclusion (Fig. 5A, B).

Fig.5

SIRT1 inhibits SC35-promoted exon 10 inclusion. SIRT1 and SC35 were expressed respectively or co-expressed in pCI/SI9-SI10 transfected HEK-293FT cells. After 48 h transfection, total RNA was extracted and the alternative splicing products of tau exon 10 were examined by RT-PCR (A) and quantitated by densitometry (B). The ratios of inclusion/exclusion of tau exon 10 were calculated and the data are represented as mean±S.E.M. (n = 3); ^*p < 0.05, ^**p < 0.01.

DISCUSSION

Proper regulation of alternative splicing of tau exon 10, generating approximately equal levels of 3R-tau and 4R-tau, is essential for maintaining normal brain function. Dysregulation of tau exon 10 splicing causes pathogenesis of tauopathies [12]. Phosphorylation of SR proteins at many serine and threonine residues regulates of tau exon 10 splicing. It has been well known that the localization and activity of SR proteins are tightly regulated by their phosphorylation levels [29 –32]. SC35 is modified by acetylation at K52 by Tip60 [16]. In this study, we demonstrate that mutation of lysine 52 to arginine leads to significant decrease in acetylation level of SC35 and reduction in tau exon 10 inclusion. These data support that post-translational modification of acetylation is also very important for the regulation of tau exon 10 splicing apart from phosphorylation.

Sirtuins play important roles in fundamental mechanisms in neurodegenerative diseases, including protein homeostasis, stress responses, neural plasticity, mitochondrial function, and inflammatory processes [20, 33]. The present study shows for the first time that SIRT1 interacts with and deacetylates SC35. Both RRM and RS domains of SC35 interacts with SIRT1. We provide the evidence that SIRT1 suppresses SC35-enhanced tau exon 10 inclusion. Decreased SIRT1 in AD brain [21] may contribute to tau pathogenesis in AD via affecting tau exon 10 splicing.

SC35 is acetylated by Tip60 and meanwhile its phosphorylation is inhibited by Tip60 indirectly [16]. Therefore, we want to know the phosphorylation status of SC35 in the presence or in the absence of SIRT1. Our results show that SIRT1 only influence the acetylation level but not the phosphorylation level of SC35, suggesting that SIRT1 inhibits the SC35-promoted tau exon 10 inclusion through changing the acetylation status of SC35. However, the exact deacetylation sites of SC35 by SIRT1 remain to be determined.

Phosphorylation is necessary for SR protein to translocate from the cytoplasm to the nucleus [34, 35] and to be recruited from nuclear speckles to nascent transcripts for splicing [36, 37]. Present data obviously shows that SIRT1 drives SC35 to locate along the periphery of the nucleus. These data suggest that similar to phosphorylation status, acetylation status is also required for the translocation of SR proteins. The mechanism and function of the translocation of SC35 impelled by SIRT1 are still need to be investigated.

Previously we found Dyrk1A phosphorylates SR proteins including ASF, SC35, SRp55, and 9G8 and regulates their function in tau exon 10 splicing [10 , 39]. SIRT1 catalyzes deacetylation of a large number of non-histone substrates in the nucleus and cytoplasm [40]. In the present study, we found that SIRT1 generally promotes tau exon 10 inclusion whereas SIRT1 inhibits the SC35-enhanced tau exon 10 inclusion. In addition to SC35, other kinds of SR proteins that is deacetylated and modulated by SIRT1 still need to be defined.

In summary, SIRT1 promotes the tau exon 10inclusion. SIRT1 interacts with and deacetylates SC35, leading to an inhibition of tau exon 10 inclusion promoted by SC35. Mutation of SC35 on lysine 52, acetylated site of Tip60, leads to the almost lostof acetylation and the reduction in tau exon 10 inclusion. Our results provide a novel insight into molecular mechanisms of the regulation of tau exon 10 splicing. These findings open new avenues for further experiments aiming at therapeutics for tauopathies.

Footnotes

ACKNOWLEDGMENTS

This work was supported in part by Nantong University and the New York State Office for People with Developmental Disabilities, and grants from National Natural Science Foundation of China (81170317 and 81473218 to WQ and 31671046 to FL), a U.S. Alzheimer’s Association Grant (DSAD-15-363172 to FL), and the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors’ disclosures available online ().

References

Grundke-Iqbal

, Iqbal

, Quinlan

, Tung

, Zaidi

, Wisniewski

(1986) Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem 261, 6084–6089.

Grundke-Iqbal

, Iqbal

, Tung

, Quinlan

, Wisniewski

, Binder

(1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci U S A 83, 4913–4917.

Spillantini

, Murrell

, Goedert

, Farlow

, Klug

, Ghetti

(1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Natl Acad Sci U S A 95, 7737–7741.

Hutton

, Lendon

, Rizzu

, Baker

, Froelich

, Houlden

, Pickering-Brown

, Chakraverty

, Isaacs

, Grover

, Hackett

, Adamson

, Lincoln

, Dickson

, Davies

, Petersen

, Stevens

, de Graaff

, Wauters

, van Baren

, Hillebrand

, Joosse

, Kwon

, Nowotny

, Che

, Norton

, Morris

, Reed

, Trojanowski

, Basun

, Lannfelt

, Neystat

, Fahn

, Dark

, Tannenberg

, Dodd

, Hayward

, Kwok

, Schofield

, Andreadis

, Snowden

, Craufurd

, Neary

, Owen

, Oostra

, Hardy

, Goate

, van Swieten

, Mann

, Lynch

, Heutink

(1998) Association of missense and 5’-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393, 702–705.

Goedert

, Spillantini

, Jakes

, Rutherford

, Crowther

(1989) Multiple isoforms of human microtubule-associated protein tau: Sequences and localization in neurofibrillary tangles of Alzheimer’s disease. Neuron 3, 519–526.

Andreadis

, Brown

, Kosik

(1992) Structure and novel exons of the human tau gene. Biochemistry 31, 10626–10633.

D’Souza

, Schellenberg

(2005) Regulation of tau isoform expression and dementia. Biochim Biophys Acta 1739, 104–115.

Yoshida

(2006) Cellular tau pathology and immunohistochemical study of tau isoforms in sporadic tauopathies. Neuropathology 26, 457–470.

Liu

, Gong

(2008) Tau exon 10 alternative splicing and tauopathies. Mol Neurodegener 3, 8.

10.

Shi

, Zhang

, Zhou

, Chohan

, Gu

, Wegiel

, Zhou

, Hwang

, Iqbal

, Grundke-Iqbal

, Gong

, Liu

(2008) Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-regulated alternative splicing of tau in Down syndrome. J Biol Chem 283, 28660–28669.

11.

Qian

, Liang

, Shi

, Jin

, Grundke-Iqbal

, Iqbal

, Gong

, Liu

(2011) Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A. Nucleic Acids Res 39, 6161–6171.

12.

Qian

, Liu

(2014) Regulation of alternative splicing of tau exon 10. Neurosci Bull 30, 367–377.

13.

Chen

, Jin

, Qian

, Liu

, Tan

, Ding

, Gu

, Iqbal

, Gong

, Zuo

, Liu

(2014) Cyclic AMP-dependentprotein kinase enhances SC35-promoted Tau exon 10 inclusion. Mol Neurobiol 49, 615–624.

14.

Alvarez

, Estivill

, de la Luna

(2003) DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly. J Cell Sci 116, 3099–3107.

15.

Takashima

(2006) GSK-3 is essential in the pathogenesis of Alzheimer’s disease. J Alzheimers Dis 9, 309–317.

16.

Edmond

, Moysan

, Khochbin

, Matthias

, Brambilla

, Gazzeri

, Eymin

(2011) Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin. EMBO J 30, 510–523.

17.

Donmez

, The neurobiology of sirtuins and their role in neurodegeneration. Trends Pharmacol Sci 33, 494–501.

18.

Michan

, Sinclair

(2007) Sirtuins in mammals: Insights into their biological function. Biochem J 404, 1–13.

19.

Haigis

, Guarente

(2006) Mammalian sirtuins–emerging roles in physiology, aging, and calorie restriction. Genes Dev 20, 2913–2921.

20.

Gan

, Mucke

(2008) Paths of convergence: Sirtuins in aging and neurodegeneration. Neuron 58, 10–14.

21.

Julien

, Tremblay

, Emond

, Lebbadi

, Salem

Jr , Bennett

, Calon

(2009) Sirtuin 1 reduction parallels the accumulation of tau in Alzheimer disease. J Neuropathol Exp Neurol 68, 48–58.

22.

Donmez

, Wang

, Cohen

, Guarente

SIRT1 suppresses beta-amyloid production by activating the alpha-secretase gene ADAM10. Cell 142, 320–332.

23.

Min

, Cho

, Zhou

, Schroeder

, Haroutunian

, Seeley

, Huang

, Shen

, Masliah

, Mukherjee

, Meyers

, Cole

, Ott

, Gan

, Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron 67, 953–966.

24.

Donmez

(2012) The effects of SIRT1 on Alzheimer’s disease models. Int J Alzheimers Dis 2012, 509529.

25.

, Guo

, Zhou

(2004) A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10. J Neurochem 90, 164–172.

26.

Mabon

, Misteli

(2005) Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo. PLoS Biol 3, e374.

27.

Kupis

, Palyga

, Tomal

, Niewiadomska

(2016) The role of sirtuins in cellular homeostasis. J Physiol Biochem 72, 371–380.

28.

, Maniatis

(1990) Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature 343, 437–441.

29.

Mermoud

, Cohen

, Lamond

(1992) Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res 20, 5263–5269.

30.

Kohtz

, Jamison

, Will

, Zuo

, Luhrmann

, Garcia-Blanco

, Manley

(1994) Protein-protein interactions and 5’-splice-site recognition in mammalian mRNA precursors. Nature 368, 119–124.

31.

Cao

, Jamison

, Garcia-Blanco

(1997) Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro. RNA 3, 1456–1467.

32.

Mermoud

, Cohen

, Lamond

(1994) Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J 13, 5679–5688.

33.

Min

, Sohn

, Cho

, Swanson

, Gan

(2013) Sirtuins in neurodegenerative diseases: An update on potential mechanisms. Front Aging Neurosci 5, 53.

34.

Stojdl

, Bell

(1999) _SR protein kinases: The splice of life. Biochem Cell Biol 77, 293–298.

35.

Lai

, Lin

, Tarn

(2001) Transportin-SR2 mediates nuclear import of phosphorylated_SR proteins. Proc Natl Acad Sci U S A 98, 10154–10159.

36.

Koizumi

, Okamoto

, Onogi

, Mayeda

, Krainer

, Hagiwara

(1999) The subcellular localization of SF2/ASF is regulated by direct interaction with_SR protein kinases (SRPKs). J Biol Chem 274, 11125–11131.

37.

Duncan

, Stojdl

, Marius

, Scheit

, Bell

(1998) The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of_SR proteins and influence pre-mRNA splicing. Exp Cell Res 241, 300–308.

38.

Ding

, Shi

, Qian

, Iqbal

, Grundke-Iqbal

, Gong

, Liu

(2012) Regulation of alternative splicing of tau exon 10 by 9G8 and Dyrk1A. Neurobiol Aging 33, 1389–1399.

39.

Yin

, Jin

, Gu

, Shi

, Zhou

, Gong

, Iqbal

, Grundke-Iqbal

, Liu

(2012) Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) modulates serine/arginine-rich protein 55 (SRp55)-promoted Tau exon 10 inclusion. J Biol Chem 287, 30497–30506.

40.

Jesko

, Wencel

, Strosznajder

(2017) Sirtuins and their roles in brain aging and neurodegenerative disorders. Neurochem Res 42, 876–890.