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Overexpression of Dyrk1A regulates cardiac troponin T splicing in cells and mice
Biochemical and Biophysical Research Communications 473 (2016) 993e998
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Overexpression of Dyrk1A regulates cardiac troponin T splicing in cells and mice Shu Lu a, Xiaomin Yin b, c, * a
Department of Intensive Care Unit, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 226001, PR China Department of Biochemistry, Medical School, Nantong University, Nantong, Jiangsu, 226001, PR China c Department of Pharmacology, Xuanwu Hospital of Capital Medical University, Beijing 100053, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 March 2016 Accepted 1 April 2016 Available online 2 April 2016
The human heart expresses four isoforms of cardiac troponin T (cTnT) through alternative splicing of exons 4 and 5 of the cTnT gene. Alternative splicing of cTnT exon 5 is developmentally regulated. cTnT isoforms containing exon 5 are expressed in the fetal and neonatal heart but not in the mature heart. SRp55 is an essential splicing factor involved in cTnT exon 5 splicing and it is phosphorylated by Dyrk1A (dual specificity tyrosine phosphorylation regulated kinase 1A). In the present study, we found Dyrk1A interacted with SRp55 and enhanced its promotion of cTnT exon 5 inclusion. The shift from cTnT exon 5 inclusion to exclusion during development was delayed in the heart of Ts65Dn mice due to Dyrk1A overexpression. These results provide new insight into the role of Dyrk1A in the neonatal cardiac development. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cardiac troponin T Exon 5 Alternative splicing Dyrk1A
1. Introduction The troponin complex is a heteromeric protein and plays an important role in the regulation of skeletal and cardiac muscle contraction. It consists of three subunits, troponin T (TnT), troponin I (TnI) and troponin C (TnC). TnT is a tropomyosin-binding subunit that regulates the interaction of the troponin complex with thin filaments [1]. TnT in cardiac muscle is expressed in forms different from those in skeletal muscle. Two isoforms of TnT are expressed in human skeletal muscle tissue, whereas several cardiac specific isoforms of TnT (cTnT) exist in human cardiac muscle. Human cTnT is encoded by the TNNT2 gene. In the human heart, alternative splicing of cTnT exons 4 and 5 generates four cTNT isoforms. cTnT1 contains both exons 4 and 5. cTnT2 and cTnT3 contain exon 5 and exon 4, respectively, and cTnT4 contains neither exon 4 nor exon 5 [2]. cTnT exon 5 splicing is conserved in several mammalian species and alternative splicing of exon 5 is under developmental regulation [3]. The isoforms of cTnT that contain exon 5 are mainly expressed in the fetal and neonatal heart, suggesting that it plays an important role in cardiac development [4]. Fetal cTnT isoforms are more sensitive to Ca2þ than adult cTnT
* Corresponding author. Department of Biochemistry, Medical school, Nantong University, Nantong, Jiangsu, 226001, PR China E-mail address: [email protected] (X. Yin). http://dx.doi.org/10.1016/j.bbrc.2016.04.004 0006-291X/© 2016 Elsevier Inc. All rights reserved.
isoforms [1,5]. Overexpression of Dyrk1A contributes to cardiac hypertrophy by inhibiting NFAT transcription factors [6]. Recently, we found that Dyrk1A phosphorylates several essential splicing factors and regulates process of pre-mRNA alternative splicing [7e10]. Dyrk1A gene locates at the Down syndrome (DS) critical region of chromosome 21. The incidence of congenital heart disease in children with DS is nearly 50% [11]. Although the underlying mechanisms remain elusive, the extra copy of human chromosome 21 in DS is thought to be responsible for the development of cardiac abnormalities. In the present study, we investigated the role of Dyrk1A in cTnT exon 5 splicing. We found that Dyrk1A promoted SRp55 modulated exon 5 inclusion. The exclusion of cTnT exon 5 during development was delayed in a Ts65Dn mouse model may be due to overexpression of Dyrk1A. 2. Materials and methods 2.1. Animals The Institutional Animal Care and Usage Committee of the Jiangsu Province approved all experiments. Ts65Dn mice (#001924) were purchased from the Jackson Laboratory. Mice were sacrificed and their heart tissues were quickly dissected out and stored
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at 80 C until use. Genotyping was carried out by quantitative real time-PCR analysis using a MX3000P qPCR machine from Stratagene (La Jolla, CA, USA). The genotype was determined by comparing the DCt values of the APP gene of each unknown sample with those of known trisomy or euploid controls. The genotyping primers and PCR program used were as the Jackson lab provided. 2.2. Plasmids, antibodies and other reagents pCEP4/SRp55, pcDNA3.0/Dyrk1A, Dyrk1AK188R and Monoclonal anti-Dyrk1A (8D9) were kindly provided by Dr. Fei Liu from New York State Institute for Basic Research. SRp55 deletion mutants were constructed as described previously [10]. Anti-HA, anti-bactin, tetramethylrhodamine isothiocyanate (TRITC)econjugated goat anti-rabbit IgG and fluorescein isothiocyanate (FITC)econjugated goat anti-mouse IgG were bought from Sigma (St. Louis, MO, USA). Anti-SRp55 was from Millipore (Billerica, MA, USA). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Pierce ECL Kit and Protein G were purchased from Thermo Fisher Scientific (Rockford, IL, USA). 2.3. Construction of pCI-neo/cTnT and DNA mutagenesis of SRp55 A human cTnT mini-gene consisting of cTnT exons 3, 4, 5, and 6 and introns 3, 4, and 5 was generated by PCR. The DNA fragment was inserted into a pCI/neo vector and was ligated by T4 ligase (Promega, WI, USA) to generate the TNNT2 mini-gene. These PCR amplification fragments were then linked through unique restriction sites at the ends of the PCR products. The final construct was cloned into a pCI-neo vector (Promega, WI, USA). The SRp55 deletion mutations were performed by amplifying part of SRp55 fulllength fragment, which were inserted into pCEP4 vector. The site mutations of SRp55 were generated with KOD PLUS kit (Toyobo, Japan) according to the manufacturer's instructions. 2.4. Co-immunoprecipitation HEK293 cells were co-transfected with pCEP4/SRp55-HA and pcDNA3/Dyrk1A for 48 h. The cells were lysed by sonication in lysate buffer (50 mM TriseHCl, pH 7.4150 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 2 mM EDTA, 1 mM PMSF, and 2 ug/ml of aprotinin, leupeptin, and pepstatin). The mouse hearts were chopped and sonicated in lysate buffer as described above. Insoluble materials from cell lysates or tissue homogenates were removed by centrifugation at 13,000 rpm for 10 min at 4 C. The extracts were incubated with anti-HA (for cultured cells) or anti-SRp55 (for mouse heart extract) overnight at 4 C. Then, protein G beads were added, and the extracts were incubated for 4 h at 4 C. After washing with lysate buffer four times and with TBS twice, the bound proteins were eluted by boiling in 2 Laemmli sample buffer for 5 min.
the cells were incubated with secondary antibodies (TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG, 1:200) with 5 mg/ml Hoechst 33342 for 15 min at room temperature. After washing with PBS, the cells were mounted with Slow Fade Gold Anti Fade reagent (Invitrogen, CA, USA) and detected with a Leica TCS-SP2 laser-scanning confocal microscope. 2.6. Western blot analysis Proteins were separated by SDS-PAGE and electronically blotted onto PVDF membranes. After blocking with 5% fat-free milk in TBS for 30 min, the membranes were incubated with primary antibody in 5% fat-free milk and 0.1% NaN3 in TBS overnight at room temperature. After washing with 0.05% Tween-20-TBS (TBST) three times, the membrane was incubated with HRP-2nd antibodies for 2 h at room temperature. Then, the membrane was washed with TBST three times and developed with ECL. 2.7. Quantitation of cTnT exon 5 splicing by reverse transcription PCR (RT-PCR) Total cellular RNA was isolated from cultured cells or mouse heart tissue using RNeasy Mini Kit (Qiagen, GmbH, Germany). Total RNA (0.5 mg) was used for first-strand cDNA synthesis with Oligo(dT)12-18 using Superscript III Reverse Transcriptase Kit (Invitrogen, CA, USA). PCR to determine the splicing products of cTnT exon 5 was performed using PrimeSTAR HS DNA Polymerase (Takara, Otsu, Shiga, Japan) with the following primer-sets: for mouse cTnT, forward: 50 -GTACGAGGAGGAACAGGAAG-30 and reverse: 50 CCAGCCTCCTCCTCCTCC-3’; for human cTnT mini-gene, forward: 50 GGTGTCCACTCCCAGTTCAA-30 and reverse: 50 -ACGCGTCGACCTTCTGCCCTGGTCTCCTCGGTCTC-30 . The PCR conditions were as follows: 30 cycles of denaturing at 98 C for 10 s, annealing at 60 C for 15 s and elongation at 72 C for 30 s. The PCR products were separated by 1.5% agarose gel or 12% native PAGE gel, visualized by ethidium bromide staining and quantitated using the Molecular Imager System (Bio-Rad, CA, USA). The ratio of PCR products cTnT E5þ/E5-was calculated. 2.8. Statistics All the graph and statistical analysis was determined by using GraphPad Prism 5.0. The data are presented as the means ± SEM. Data points were compared by the unpaired two-tailed Student's t test or one-way ANOVA analysis. For all analyses, p < 0.05 indicated a statistically significant difference. 3. Results 3.1. Exon 5 in the pCI/cTnTE3-E6 human mini-cTnT gene is alternatively spliced
2.5. Co-localization study HepG2 and H9C2 cells were plated on 24-well plates with coated coverslips one day prior to transfection at ~30% confluence. HA-tagged SRp55 was co-expressed with Dyrk1A in HepG2 cells as described above. 48 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and blocked with 10% goat serum in 0.2% Triton X-100-PBS for 1 h at 37 C. Then, the cells were incubated with rabbit anti-HA antibody (1:1000) and mouse antiDyrk1A (8D9, 1:2000) overnight at 4 C. H9C2 cells were used to study the co-localization of endogenous SRp55 and Dyrk1A with mouse anti-SRp55 (1:1000) and rabbit anti-Dyrk1A (1:1000). Then,
To investigate the regulation of cTnT exon 5 splicing, we constructed pCI/cTnTE3-6, a cTnT mini-gene, which contains exons 3, 4, 5, 6 and introns 3, 4, 5 (Fig. 1A). To study whether exon 5 is able to be alternatively spliced, we transfected pCI/cTnTE3-E6 into HEK293 cells and measured its splicing products by RT-PCR 48 h after transfection. We observed two major bands at ~200 bp (Fig. 1B). Sequencing of these two PCR products revealed that the upper band included exons 3, 4, 5, and 6, whereas the lower band only contained exons 3, 4, and 6 (Fig. 1B). This result suggests that exon 5 in the cTnT mini-gene is alternatively spliced and pCI/cTnTE3-E6 is an adequate tool for the study the alternative splicing of cTnT exon 5.
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SRp55 contains two RNA recognition (RRM) motifs at N-terminus and two RS domains at C-terminus, the phosphorylation level of latter is responsible for its splicing activity. To investigate the domain-specific role of SRp55, we made various deletion mutants of C-terminal SRp55 (Fig. 2C) and overexpressed them together with pCI/cTnTE3-E6 in HEK293 cells. We found that deletion of RS2 domain did not affect SRp55 function (Fig. 2D). However, the further deletion of Proline-rich domain or whole RS domain, led to the failure of SRp55 to promote cTnT exon 5 inclusion (Fig. 2D). These data suggest that SRp55 requires RS1 and proline-rich domains to promote cTnT exon 5 inclusion. 3.3. Dyrk1A interacts with SRp55 in mammalian cells and in mouse hearts
Fig. 1. cTnT exon 5 is alternatively spliced in pCI/cTnTE3-E6 mini-gene. A. Schematic of cTnT isoforms generated from the alternative splicing of exons 4 and 5. B. Alternative splicing of exon 5 in pCI/cTnTE3-E6 mini-gene. pCI/cTnTE3-E6, composing of exons 3, 4, 5, and 6 and introns 3, 4, and 5, was transfected into HEK293 cells. The splicing products were determined by RT-PCR 48 h after transfection and confirmed by sequencing.
3.2. SRp55 promotes cTnT exon 5 inclusion A study using UV cross-linking and immunoprecipitation indicated that SRp55 participates in the regulation of alternative splicing of cTnT exon 5 [12]. Here we co-transfected pCI/cTnTE3-E6 with pCEP4/SRp55 or siRNA of SRp55 into HEK-293FT cells and then measured the alternative splicing of cTnT exon 5. We found that the overexpression of SRp55 promoted cTnT exon 5 inclusion, whereas the knockdown of SRp55 suppressed exon 5 inclusion (Fig. 2A). We also studied the effect of SRp55 on cTnT exon 5 splicing in three different types of cells, HEK293-FT, SH-SY5Y and HeLa cells. We found that SRp55 increased cTnT exon 5 inclusion in all three mammalian cell lines (Fig. 2B).
Dyrk1A plays a role in regulating the biogenesis of the splicing speckle compartmentis and is ubiquitously expressed with high levels in the heart [13]. To study the interaction between Dyrk1A and SRp55, we performed a co-immunoprecipitation assay. We found that Dyrk1A was co-immunoprecipitated with SRp55 in HEK293 cells overexpressing HA tagged SRp55 (Fig. 3A). Next, to determine the interaction of endogenous SRp55 and Dyrk1A, we incubated the crude extracts of mouse heart muscle tissue with anti-SRp55 and then studied whether Dyrk1A could be coimmunoprecipitated. We found a significant amount of Dyrk1A in the immunoprecipitates after treatment with anti-SRp55 (Fig. 3B), suggesting that Dyrk1A interacts with SRp55 in cardiac cells as in the cultured HEK293 cells. Furthermore, we found that SRp55 and Dyrk1A co-localized well in the nuclei of in HepG2 and H9C2 (Fig. 3C and 3D). 3.4. Dyrk1A modulates SRp55-promoted cTnT exon 5 inclusion To detect whether Dyrk1A affects SRp55-promoted cTnT exon 5
Fig. 2. SRp55 promotes cTnT exon 5 inclusion. A. SRp55 promoted cTnT exon 5 inclusion. HEK293FT cells were co-transfected with pCI/cTnTE3-E6 and SRp55 or SRp55 siRNA for 48 h. B. SRp55 promoted cTnT exon 5 in different types of cells. HEK293FT, SH-SY5Y and HeLa cells were co-transfected with pCI/cTnTE3-E6and SRp55. C. Schematic structures of SRp55 deletion mutants. D. C-terminal domain of SRp55 was required for promoting cTnT exon 5 inclusion. HEK293 cells were co-transfected pCI/cTnTE3-E6 with pCEP4/SRp55 or its deletion mutants for 48 h. The splicing products of cTnT exon 5 were measured by RT-PCR and the ratios of inclusion/exclusion of cTnT exon 5 are presented as the mean ± SEM. **, p < 0.01, as compared to mock transfection; ##, p < 0.01 as compared to SRp55FL.
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Fig. 3. Dyrk1A interacts with SRp55 and regulates SRp55 promoted cTnT exon 5 inclusion. A. Dyrk1A and HA-SRp55 were co-overexpressed in HEK293-FT cells for 48h. SRp55 was immunoprecipitated by anti-HA and analyzed by Western blots developed with anti-HA (for SRp55) and anti-Dyrk1A. B. Interaction of endogenous SRp55 and Dyrk1A. SRp55 was immunoprecipitated from mouse heart muscle extract with anti-SRp55 and analyzed by Western blots C. Co-localization of SRp55 and Dyrk1A in HepG2 cells. HepG2 cells were overexpressed with SRp55 tagged with HA and Dyrk1A. The cells were immunostained with anti-HA or 8D9 D. Co-localization of endogenous SRp55 and Dyrk1A in H9C2 cells. H9C2 cells were immunostained with anti-SRp55 or 8D9. E. HEK293FT cells were co-transfected with pCI/cTnTE3-E6, pCEP4/SRp55 and pcDNA3/Dyrk1A or pcDNA/Dyrk1AK188R. F. HEK293 cells were co-transfected with pCI/cTnTE3-E6, pCEP4/SRp55 or its site mutated forms. The alternative splicing products of cTnT exon 5 were measured by RT-PCR. The ratios of cTnT exon 5 inclusion (E5þ) to exclusion (E5-) in panel E and F were calculated and presented as mean ± SEM.*, p < 0.05, via to mock; ##, p < 0.01; ###, p < 0.001, compared with SRp55FL group; &, p < 0.05; &&, p < 0.01 compared with Dyrk1A or Dyrk1AK188R groups.
inclusion, we transfected pCI/cTnTE3-E6 into HEK293FT cells together with pCEP4/SRp55 or/and pcDNA3/Dyrk1A. We observed
that the expression of either SRp55 or Dyrk1A increased cTnT inclusion. Co-overexpression of SRp55 and Dyrk1A showed a
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synergetic effect on the promotion of cTnT exon 5 inclusion (Fig. 3E). These results suggest that Dyrk1A enhances SRp55promoted cTnT exon 5 inclusion. However, the overexpression of the dead enzyme form of Dyrk1A, Dyrk1AK188R, had no effect on cTnT exon 5 splicing (Fig. 3E), indicating that the kinase activity of Dyrk1A is essential for its role in the regulation of cTnT splicing. SRp55 has three putative Dyrk1A sites, Ser-280, Ser-303, and Ser-316. To know which sites may mediate the role of Dyrk1A on SRp55-promoted cTnT exon 5 inclusion, we mutated these three sites to Alanine and determined their effects. We found that mutation of each sites decreased the cTnT exon 5 inclusion. Among the three sites, SRp55S280A had the strongest suppression effect in cTnT exon 5 inclusion (Fig. 3F). These results supported the finding that phosphorylation of SRp55 by Dyrk1A promotes cTnT exon 5 inclusion.
3.5. cTnT exon 5 inclusion is increased in the heart of Ts65Dn mice Alternative splicing of cTnT exon 5 is regulated developmentally. cTnT isoforms with exon 5 are expressed mainly in the pre- and neonatal stages, and gradually cTnT isoforms with exon 5 stop being expressed after birth [3,18]. To investigate the impact of Dyrk1A overexpression on cTnT exon 5 splicing in vivo, we determined the splicing products of cTnT exon 5 in the neonatal heart of Ts65Dn mice, a commonly used mouse model of DS in which Dyrk1A is overexpressed. As expected, we observed an approximate increase of 50% in the level of Dyrk1A in the heart tissue of Ts65Dn mice compared to their wild type littermates (Fig. 4A and 4B). The neonatal hearts expressed four isoforms of cTnT by alternative splicing of exons 4 and 5 [14]. Isoforms of cTnT with exon 5 were expressed in P5 and decreased during later development (Fig. 4C). Compared to the wild type littermates, the shift from cTnT exon 5 inclusion to exclusion was delayed obviously at around P10 in Ts65Dn hearts (Fig. 4C). To confirm this observation, we further determined cTnT exon 5 inclusion/exclusion in the hearts from Ts65Dn littermates at P10 by running PAGE gels to separated the cTnT isoform bands. We found that the ratio of cTnT E5þ/E5-in the
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Ts65Dn mice was ~1.5-fold higher than that of the control mice (Fig. 4D). Thus, overexpression of Dyrk1A promotes exon 5 inclusion in vivo and may cause a delay in exon 5 exclusion during neonatal cardiac development.
4. Discussion cTnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration. Transcripts for this gene undergo alternative splicing, which results in many tissue-specific isoforms [15]; however, the full-length nature of some of these variants has not yet been determined. Mutations to TNNT2 may be associated with mild or absent hypertrophy and predominant restrictive disease, with a high risk of sudden cardiac death [16]. Advancement to dilated cardiomyopathy may be more rapid in patients with TNNT2 mutations than in those with myosin heavy chain mutations [17,18]. No mutations have been shown to occur in the N- terminus of human cTnT. However, Biesiadecki et al. reported that cTnT N-terminal exons in several mammals undergo aberrant splicing resulting in DCM [19], indicating that abnormalities in cTnT mRNA splicing may play a role in the pathogenesis of congenital heart disease. The exclusion of exon 4 from adult human cTnT is responsible for the low molecular weight of TnT4 found in hypertrophic and failing hearts [2]. The alternative splicing of exon 5 in cTnT is tightly regulated during development such that it is normally excluded from all adult cTnT [3]. By analyzing Ts65Dn transgenic mouse hearts, we found that the expression of embryonic cTnT including exon 5 was increased (Fig. 4D). This was positively correlated with a slight reduction in Ts65Dn mouse heart weight (data not shown). These data support the notion that the overexpression of Dyrk1A in Ts65Dn mouse hearts may have a negative effect on cardiac development. Therefore, it may constitute a pathogenic factor in the development of congenital heart disease. Congenital heart disease occurs in 41%e56% of all DS patients
Fig. 4. Developmental switch off of cTnT exon 5 is delayed in Ts65Dn mice. A. Dyrk1A was overexpressed in the hearts from Ts65Dn mice. Homogenates from mouse hearts at P5, P10, P15 were analyzed by Western blots developed with anti-Dyrk1A and anti-b-actin. B. Quantifications of the blots after normalization with b-actin are presented as mean ± S.E.M. in the graph. C. Developmental switch off of cTnT exon 5 was delayed in Ts65Dn at P10. Total RNA of the mouse hearts from P5, P10, and P15 were extracted and then subjected to RT-PCR. D. The ratio of cTnT isoforms with and without exon 5 was increased in Ts65Dn. The alternative splicing of cTnT exon 5 in mouse hearts at P10 was measured byRT-PCR and separated by PAGE gel. The relative ratio of cTnT isoforms with exon 5 (E5þ) and without exon 5 (E5-) was calculated and the data were presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
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[20e23]. Analyses of the expression profiles of individual trisomic genes responsible for the DS phenotype have been performed in recent years [24,25]. Heart defects were correlated with the overexpression of 20 genes in mouse models of this human genetic disease [26]. Among these genes, Dyrk1A has been proposed as a causative gene for DS associated heart defects. Our group has reported that Dyrk1A is an important SR protein kinase and is involved in regulating tau pre-mRNA splicing [7e10]. Here, we found that its overexpression in mammalian cells and Ts65Dn mouse hearts promoted cTnT exon 5 splicing, providing evidence for its role in pre-mRNA alternative splicing. We previously found that Dyrk1A interacts with SRp55 through RRM domains and phosphorylates SRp55 mainly with RS1 and prorich domains [10]. In this study, the interaction of Dyrk1A with SRp55 was confirmed in cardiac cells and was shown to promote cTnT exon 5 inclusion. The role of Dyrk1A in SRp55-mediated cTnT exon 5 inclusion is SRp55 C-terminus dependent (Fig. 2D), suggesting that Dyrk1A may modulate SRp55 function by phosphorylating SRp55 at C terminus. By phosphorylation of SRp55 at Dyrk1A favorite site Ser-280, Dyrk1A enhanced SRp55's splicing activity and causes an increase in cTnT E5þ isoforms. In conclusion, we found that Dyrk1A regulates SRp55modulated cTnT exon 5 splicing. We identified the novel splicing events in Ts65Dn mice during neonatal cardiac development. These changes in cTnT exon 5 splicing may contribute to the pathogenesis of heart disorders in DS patients. Acknowledgments This work was supported in part by funds from Natural Science Foundation of Nantong University (14ZY020); National Natural Science Foundation of China Grant (grant 81503077); Basic Research Program of Jiangsu Education Department Grant (grant 13KJB320015); and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD); The Open Program of Key Laboratory Foundation of Capital Medical University (1300-1140170555).
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Transparency document [20]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.004
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development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart, J. Biol. Chem. 279 (2004) 49579e49587. C. Kuhn, D. Frank, R. Will, C. Jaschinski, R. Frauen, H.A. Katus, N. Frey, DYRK1A is a novel negative regulator of cardiomyocyte hypertrophy, J. Biol. Chem. 284 (2009) 17320e17327. J. Shi, T. Zhang, C. Zhou, M.O. Chohan, X. Gu, J. Wegiel, J. Zhou, Y.W. Hwang, K. Iqbal, I. Grundke-Iqbal, C.X. Gong, F. Liu, Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-regulated alternative splicing of tau in Down syndrome, J. Biol. Chem. 283 (2008) 28660e28669. S. Ding, J. Shi, W. Qian, K. Iqbal, I. Grundke-Iqbal, C.X. Gong, and F. Liu, Regulation of alternative splicing of tau exon 10 by 9G8 and Dyrk1A. Neurobiol. Aging 33 1389e1399. W. Qian, H. Liang, J. Shi, N. Jin, I. Grundke-Iqbal, K. Iqbal, C.X. Gong, and F. Liu, Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A. Nucleic Acids Res. 39 6161e6171. X. Yin, N. Jin, J. Gu, J. Shi, J. Zhou, C.X. Gong, K. Iqbal, I. Grundke-Iqbal, and F. Liu, Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) modulates serine/arginine-rich protein 55 (SRp55)-promoted Tau exon 10 inclusion. J. Biol. Chem. 287 30497e30506. Q. Yang, S.A. Rasmussen, J.M. Friedman, Mortality associated with Down's syndrome in the USA from 1983 to 1997: a population-based study, Lancet 359 (2002) 1019e1025. J. Ramchatesingh, A.M. Zahler, K.M. Neugebauer, M.B. Roth, T.A. Cooper, A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer, Mol. Cell. Biol. 15 (1995) 4898e4907. M. Okui, T. Ide, K. Morita, E. Funakoshi, F. Ito, K. Ogita, Y. Yoneda, J. Kudoh, N. Shimizu, High-level expression of the Mnb/Dyrk1A gene in brain and heart during rat early development, Genomics 62 (1999) 165e171. X. Xu, D. Yang, J.H. Ding, W. Wang, P.H. Chu, N.D. Dalton, H.Y. Wang, J.R. Bermingham Jr., Z. Ye, F. Liu, M.G. Rosenfeld, J.L. Manley, J. Ross Jr., J. Chen, R.P. Xiao, H. Cheng, X.D. Fu, ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle, Cell 120 (2005) 59e72. R.E. Breitbart, H.T. Nguyen, R.M. Medford, A.T. Destree, V. Mahdavi, B. NadalGinard, Intricate combinatorial patterns of exon splicing generate multiple regulated troponin T isoforms from a single gene, Cell 41 (1985) 67e82. A.M. Varnava, P.M. Elliott, C. Baboonian, F. Davison, M.J. Davies, W.J. McKenna, Hypertrophic cardiomyopathy: histopathological features of sudden death in cardiac troponin T disease, Circulation 104 (2001) 1380e1384. N. Fujino, M. Shimizu, H. Ino, K. Okeie, M. Yamaguchi, T. Yasuda, H. Kokado, H. Mabuchi, Cardiac troponin T Arg92Trp mutation and progression from hypertrophic to dilated cardiomyopathy, Clin. Cardiol. 24 (2001) 397e402. N. Fujino, M. Shimizu, H. Ino, M. Yamaguchi, T. Yasuda, M. Nagata, T. Konno, H. Mabuchi, A novel mutation Lys273Glu in the cardiac troponin T gene shows high degree of penetrance and transition from hypertrophic to dilated cardiomyopathy, Am. J. Cardiol. 89 (2002) 29e33. B.J. Biesiadecki, B.D. Elder, Z.B. Yu, J.P. Jin, Cardiac troponin T variants produced by aberrant splicing of multiple exons in animals with high instances of dilated cardiomyopathy, J. Biol. Chem. 277 (2002) 50275e50285. S.B. Freeman, L.F. Taft, K.J. Dooley, K. Allran, S.L. Sherman, T.J. Hassold, M.J. Khoury, D.M. Saker, Population-based study of congenital heart defects in down syndrome, Am. J. Med. Genet. 80 (1998) 213e217. S.B. Freeman, L.H. Bean, E.G. Allen, S.W. Tinker, A.E. Locke, C. Druschel, C.A. Hobbs, P.A. Romitti, M.H. Royle, C.P. Torfs, K.J. Dooley, S.L. Sherman, Ethnicity, sex, and the incidence of congenital heart defects: a report from the national down syndrome project, Genet. Med. 10 (2008) 173e180. C. Stoll, Y. Alembik, B. Dott, M.P. Roth, Study of Down syndrome in 238,942 consecutive births, Ann. Genet. 41 (1998) 44e51. C.P. Torfs, R.E. Christianson, Anomalies in Down syndrome individuals in a large population-based registry, Am. J. Med. Genet. 77 (1998) 431e438. P. Kahlem, M. Sultan, R. Herwig, M. Steinfath, D. Balzereit, B. Eppens, N.G. Saran, M.T. Pletcher, S.T. South, G. Stetten, H. Lehrach, R.H. Reeves, M.L. Yaspo, Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome, Genome Res. 14 (2004) 1258e1267. R. Lyle, C. Gehrig, C. Neergaard-Henrichsen, S. Deutsch, S.E. Antonarakis, Gene expression from the aneuploid chromosome in a trisomy mouse model of down syndrome, Genome Res. 14 (2004) 1268e1274. C. Liu, M. Morishima, T. Yu, S. Matsui, L. Zhang, D. Fu, A. Pao, A.C. Costa, K.J. Gardiner, J.K. Cowell, N.J. Nowak, M.S. Parmacek, P. Liang, A. Baldini, and Y.E. Yu, Genetic analysis of Down syndrome-associated heart defects in mice. Hum. Genet. 130 623e632.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Overexpression of Dyrk1A regulates cardiac troponin T splicing in cells and mice Shu Lu a, Xiaomin Yin b, c, * a
Department of Intensive Care Unit, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 226001, PR China Department of Biochemistry, Medical School, Nantong University, Nantong, Jiangsu, 226001, PR China c Department of Pharmacology, Xuanwu Hospital of Capital Medical University, Beijing 100053, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 March 2016 Accepted 1 April 2016 Available online 2 April 2016
The human heart expresses four isoforms of cardiac troponin T (cTnT) through alternative splicing of exons 4 and 5 of the cTnT gene. Alternative splicing of cTnT exon 5 is developmentally regulated. cTnT isoforms containing exon 5 are expressed in the fetal and neonatal heart but not in the mature heart. SRp55 is an essential splicing factor involved in cTnT exon 5 splicing and it is phosphorylated by Dyrk1A (dual specificity tyrosine phosphorylation regulated kinase 1A). In the present study, we found Dyrk1A interacted with SRp55 and enhanced its promotion of cTnT exon 5 inclusion. The shift from cTnT exon 5 inclusion to exclusion during development was delayed in the heart of Ts65Dn mice due to Dyrk1A overexpression. These results provide new insight into the role of Dyrk1A in the neonatal cardiac development. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cardiac troponin T Exon 5 Alternative splicing Dyrk1A
1. Introduction The troponin complex is a heteromeric protein and plays an important role in the regulation of skeletal and cardiac muscle contraction. It consists of three subunits, troponin T (TnT), troponin I (TnI) and troponin C (TnC). TnT is a tropomyosin-binding subunit that regulates the interaction of the troponin complex with thin filaments [1]. TnT in cardiac muscle is expressed in forms different from those in skeletal muscle. Two isoforms of TnT are expressed in human skeletal muscle tissue, whereas several cardiac specific isoforms of TnT (cTnT) exist in human cardiac muscle. Human cTnT is encoded by the TNNT2 gene. In the human heart, alternative splicing of cTnT exons 4 and 5 generates four cTNT isoforms. cTnT1 contains both exons 4 and 5. cTnT2 and cTnT3 contain exon 5 and exon 4, respectively, and cTnT4 contains neither exon 4 nor exon 5 [2]. cTnT exon 5 splicing is conserved in several mammalian species and alternative splicing of exon 5 is under developmental regulation [3]. The isoforms of cTnT that contain exon 5 are mainly expressed in the fetal and neonatal heart, suggesting that it plays an important role in cardiac development [4]. Fetal cTnT isoforms are more sensitive to Ca2þ than adult cTnT
* Corresponding author. Department of Biochemistry, Medical school, Nantong University, Nantong, Jiangsu, 226001, PR China E-mail address: [email protected] (X. Yin). http://dx.doi.org/10.1016/j.bbrc.2016.04.004 0006-291X/© 2016 Elsevier Inc. All rights reserved.
isoforms [1,5]. Overexpression of Dyrk1A contributes to cardiac hypertrophy by inhibiting NFAT transcription factors [6]. Recently, we found that Dyrk1A phosphorylates several essential splicing factors and regulates process of pre-mRNA alternative splicing [7e10]. Dyrk1A gene locates at the Down syndrome (DS) critical region of chromosome 21. The incidence of congenital heart disease in children with DS is nearly 50% [11]. Although the underlying mechanisms remain elusive, the extra copy of human chromosome 21 in DS is thought to be responsible for the development of cardiac abnormalities. In the present study, we investigated the role of Dyrk1A in cTnT exon 5 splicing. We found that Dyrk1A promoted SRp55 modulated exon 5 inclusion. The exclusion of cTnT exon 5 during development was delayed in a Ts65Dn mouse model may be due to overexpression of Dyrk1A. 2. Materials and methods 2.1. Animals The Institutional Animal Care and Usage Committee of the Jiangsu Province approved all experiments. Ts65Dn mice (#001924) were purchased from the Jackson Laboratory. Mice were sacrificed and their heart tissues were quickly dissected out and stored
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at 80 C until use. Genotyping was carried out by quantitative real time-PCR analysis using a MX3000P qPCR machine from Stratagene (La Jolla, CA, USA). The genotype was determined by comparing the DCt values of the APP gene of each unknown sample with those of known trisomy or euploid controls. The genotyping primers and PCR program used were as the Jackson lab provided. 2.2. Plasmids, antibodies and other reagents pCEP4/SRp55, pcDNA3.0/Dyrk1A, Dyrk1AK188R and Monoclonal anti-Dyrk1A (8D9) were kindly provided by Dr. Fei Liu from New York State Institute for Basic Research. SRp55 deletion mutants were constructed as described previously [10]. Anti-HA, anti-bactin, tetramethylrhodamine isothiocyanate (TRITC)econjugated goat anti-rabbit IgG and fluorescein isothiocyanate (FITC)econjugated goat anti-mouse IgG were bought from Sigma (St. Louis, MO, USA). Anti-SRp55 was from Millipore (Billerica, MA, USA). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Pierce ECL Kit and Protein G were purchased from Thermo Fisher Scientific (Rockford, IL, USA). 2.3. Construction of pCI-neo/cTnT and DNA mutagenesis of SRp55 A human cTnT mini-gene consisting of cTnT exons 3, 4, 5, and 6 and introns 3, 4, and 5 was generated by PCR. The DNA fragment was inserted into a pCI/neo vector and was ligated by T4 ligase (Promega, WI, USA) to generate the TNNT2 mini-gene. These PCR amplification fragments were then linked through unique restriction sites at the ends of the PCR products. The final construct was cloned into a pCI-neo vector (Promega, WI, USA). The SRp55 deletion mutations were performed by amplifying part of SRp55 fulllength fragment, which were inserted into pCEP4 vector. The site mutations of SRp55 were generated with KOD PLUS kit (Toyobo, Japan) according to the manufacturer's instructions. 2.4. Co-immunoprecipitation HEK293 cells were co-transfected with pCEP4/SRp55-HA and pcDNA3/Dyrk1A for 48 h. The cells were lysed by sonication in lysate buffer (50 mM TriseHCl, pH 7.4150 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 2 mM EDTA, 1 mM PMSF, and 2 ug/ml of aprotinin, leupeptin, and pepstatin). The mouse hearts were chopped and sonicated in lysate buffer as described above. Insoluble materials from cell lysates or tissue homogenates were removed by centrifugation at 13,000 rpm for 10 min at 4 C. The extracts were incubated with anti-HA (for cultured cells) or anti-SRp55 (for mouse heart extract) overnight at 4 C. Then, protein G beads were added, and the extracts were incubated for 4 h at 4 C. After washing with lysate buffer four times and with TBS twice, the bound proteins were eluted by boiling in 2 Laemmli sample buffer for 5 min.
the cells were incubated with secondary antibodies (TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG, 1:200) with 5 mg/ml Hoechst 33342 for 15 min at room temperature. After washing with PBS, the cells were mounted with Slow Fade Gold Anti Fade reagent (Invitrogen, CA, USA) and detected with a Leica TCS-SP2 laser-scanning confocal microscope. 2.6. Western blot analysis Proteins were separated by SDS-PAGE and electronically blotted onto PVDF membranes. After blocking with 5% fat-free milk in TBS for 30 min, the membranes were incubated with primary antibody in 5% fat-free milk and 0.1% NaN3 in TBS overnight at room temperature. After washing with 0.05% Tween-20-TBS (TBST) three times, the membrane was incubated with HRP-2nd antibodies for 2 h at room temperature. Then, the membrane was washed with TBST three times and developed with ECL. 2.7. Quantitation of cTnT exon 5 splicing by reverse transcription PCR (RT-PCR) Total cellular RNA was isolated from cultured cells or mouse heart tissue using RNeasy Mini Kit (Qiagen, GmbH, Germany). Total RNA (0.5 mg) was used for first-strand cDNA synthesis with Oligo(dT)12-18 using Superscript III Reverse Transcriptase Kit (Invitrogen, CA, USA). PCR to determine the splicing products of cTnT exon 5 was performed using PrimeSTAR HS DNA Polymerase (Takara, Otsu, Shiga, Japan) with the following primer-sets: for mouse cTnT, forward: 50 -GTACGAGGAGGAACAGGAAG-30 and reverse: 50 CCAGCCTCCTCCTCCTCC-3’; for human cTnT mini-gene, forward: 50 GGTGTCCACTCCCAGTTCAA-30 and reverse: 50 -ACGCGTCGACCTTCTGCCCTGGTCTCCTCGGTCTC-30 . The PCR conditions were as follows: 30 cycles of denaturing at 98 C for 10 s, annealing at 60 C for 15 s and elongation at 72 C for 30 s. The PCR products were separated by 1.5% agarose gel or 12% native PAGE gel, visualized by ethidium bromide staining and quantitated using the Molecular Imager System (Bio-Rad, CA, USA). The ratio of PCR products cTnT E5þ/E5-was calculated. 2.8. Statistics All the graph and statistical analysis was determined by using GraphPad Prism 5.0. The data are presented as the means ± SEM. Data points were compared by the unpaired two-tailed Student's t test or one-way ANOVA analysis. For all analyses, p < 0.05 indicated a statistically significant difference. 3. Results 3.1. Exon 5 in the pCI/cTnTE3-E6 human mini-cTnT gene is alternatively spliced
2.5. Co-localization study HepG2 and H9C2 cells were plated on 24-well plates with coated coverslips one day prior to transfection at ~30% confluence. HA-tagged SRp55 was co-expressed with Dyrk1A in HepG2 cells as described above. 48 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and blocked with 10% goat serum in 0.2% Triton X-100-PBS for 1 h at 37 C. Then, the cells were incubated with rabbit anti-HA antibody (1:1000) and mouse antiDyrk1A (8D9, 1:2000) overnight at 4 C. H9C2 cells were used to study the co-localization of endogenous SRp55 and Dyrk1A with mouse anti-SRp55 (1:1000) and rabbit anti-Dyrk1A (1:1000). Then,
To investigate the regulation of cTnT exon 5 splicing, we constructed pCI/cTnTE3-6, a cTnT mini-gene, which contains exons 3, 4, 5, 6 and introns 3, 4, 5 (Fig. 1A). To study whether exon 5 is able to be alternatively spliced, we transfected pCI/cTnTE3-E6 into HEK293 cells and measured its splicing products by RT-PCR 48 h after transfection. We observed two major bands at ~200 bp (Fig. 1B). Sequencing of these two PCR products revealed that the upper band included exons 3, 4, 5, and 6, whereas the lower band only contained exons 3, 4, and 6 (Fig. 1B). This result suggests that exon 5 in the cTnT mini-gene is alternatively spliced and pCI/cTnTE3-E6 is an adequate tool for the study the alternative splicing of cTnT exon 5.
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SRp55 contains two RNA recognition (RRM) motifs at N-terminus and two RS domains at C-terminus, the phosphorylation level of latter is responsible for its splicing activity. To investigate the domain-specific role of SRp55, we made various deletion mutants of C-terminal SRp55 (Fig. 2C) and overexpressed them together with pCI/cTnTE3-E6 in HEK293 cells. We found that deletion of RS2 domain did not affect SRp55 function (Fig. 2D). However, the further deletion of Proline-rich domain or whole RS domain, led to the failure of SRp55 to promote cTnT exon 5 inclusion (Fig. 2D). These data suggest that SRp55 requires RS1 and proline-rich domains to promote cTnT exon 5 inclusion. 3.3. Dyrk1A interacts with SRp55 in mammalian cells and in mouse hearts
Fig. 1. cTnT exon 5 is alternatively spliced in pCI/cTnTE3-E6 mini-gene. A. Schematic of cTnT isoforms generated from the alternative splicing of exons 4 and 5. B. Alternative splicing of exon 5 in pCI/cTnTE3-E6 mini-gene. pCI/cTnTE3-E6, composing of exons 3, 4, 5, and 6 and introns 3, 4, and 5, was transfected into HEK293 cells. The splicing products were determined by RT-PCR 48 h after transfection and confirmed by sequencing.
3.2. SRp55 promotes cTnT exon 5 inclusion A study using UV cross-linking and immunoprecipitation indicated that SRp55 participates in the regulation of alternative splicing of cTnT exon 5 [12]. Here we co-transfected pCI/cTnTE3-E6 with pCEP4/SRp55 or siRNA of SRp55 into HEK-293FT cells and then measured the alternative splicing of cTnT exon 5. We found that the overexpression of SRp55 promoted cTnT exon 5 inclusion, whereas the knockdown of SRp55 suppressed exon 5 inclusion (Fig. 2A). We also studied the effect of SRp55 on cTnT exon 5 splicing in three different types of cells, HEK293-FT, SH-SY5Y and HeLa cells. We found that SRp55 increased cTnT exon 5 inclusion in all three mammalian cell lines (Fig. 2B).
Dyrk1A plays a role in regulating the biogenesis of the splicing speckle compartmentis and is ubiquitously expressed with high levels in the heart [13]. To study the interaction between Dyrk1A and SRp55, we performed a co-immunoprecipitation assay. We found that Dyrk1A was co-immunoprecipitated with SRp55 in HEK293 cells overexpressing HA tagged SRp55 (Fig. 3A). Next, to determine the interaction of endogenous SRp55 and Dyrk1A, we incubated the crude extracts of mouse heart muscle tissue with anti-SRp55 and then studied whether Dyrk1A could be coimmunoprecipitated. We found a significant amount of Dyrk1A in the immunoprecipitates after treatment with anti-SRp55 (Fig. 3B), suggesting that Dyrk1A interacts with SRp55 in cardiac cells as in the cultured HEK293 cells. Furthermore, we found that SRp55 and Dyrk1A co-localized well in the nuclei of in HepG2 and H9C2 (Fig. 3C and 3D). 3.4. Dyrk1A modulates SRp55-promoted cTnT exon 5 inclusion To detect whether Dyrk1A affects SRp55-promoted cTnT exon 5
Fig. 2. SRp55 promotes cTnT exon 5 inclusion. A. SRp55 promoted cTnT exon 5 inclusion. HEK293FT cells were co-transfected with pCI/cTnTE3-E6 and SRp55 or SRp55 siRNA for 48 h. B. SRp55 promoted cTnT exon 5 in different types of cells. HEK293FT, SH-SY5Y and HeLa cells were co-transfected with pCI/cTnTE3-E6and SRp55. C. Schematic structures of SRp55 deletion mutants. D. C-terminal domain of SRp55 was required for promoting cTnT exon 5 inclusion. HEK293 cells were co-transfected pCI/cTnTE3-E6 with pCEP4/SRp55 or its deletion mutants for 48 h. The splicing products of cTnT exon 5 were measured by RT-PCR and the ratios of inclusion/exclusion of cTnT exon 5 are presented as the mean ± SEM. **, p < 0.01, as compared to mock transfection; ##, p < 0.01 as compared to SRp55FL.
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Fig. 3. Dyrk1A interacts with SRp55 and regulates SRp55 promoted cTnT exon 5 inclusion. A. Dyrk1A and HA-SRp55 were co-overexpressed in HEK293-FT cells for 48h. SRp55 was immunoprecipitated by anti-HA and analyzed by Western blots developed with anti-HA (for SRp55) and anti-Dyrk1A. B. Interaction of endogenous SRp55 and Dyrk1A. SRp55 was immunoprecipitated from mouse heart muscle extract with anti-SRp55 and analyzed by Western blots C. Co-localization of SRp55 and Dyrk1A in HepG2 cells. HepG2 cells were overexpressed with SRp55 tagged with HA and Dyrk1A. The cells were immunostained with anti-HA or 8D9 D. Co-localization of endogenous SRp55 and Dyrk1A in H9C2 cells. H9C2 cells were immunostained with anti-SRp55 or 8D9. E. HEK293FT cells were co-transfected with pCI/cTnTE3-E6, pCEP4/SRp55 and pcDNA3/Dyrk1A or pcDNA/Dyrk1AK188R. F. HEK293 cells were co-transfected with pCI/cTnTE3-E6, pCEP4/SRp55 or its site mutated forms. The alternative splicing products of cTnT exon 5 were measured by RT-PCR. The ratios of cTnT exon 5 inclusion (E5þ) to exclusion (E5-) in panel E and F were calculated and presented as mean ± SEM.*, p < 0.05, via to mock; ##, p < 0.01; ###, p < 0.001, compared with SRp55FL group; &, p < 0.05; &&, p < 0.01 compared with Dyrk1A or Dyrk1AK188R groups.
inclusion, we transfected pCI/cTnTE3-E6 into HEK293FT cells together with pCEP4/SRp55 or/and pcDNA3/Dyrk1A. We observed
that the expression of either SRp55 or Dyrk1A increased cTnT inclusion. Co-overexpression of SRp55 and Dyrk1A showed a
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synergetic effect on the promotion of cTnT exon 5 inclusion (Fig. 3E). These results suggest that Dyrk1A enhances SRp55promoted cTnT exon 5 inclusion. However, the overexpression of the dead enzyme form of Dyrk1A, Dyrk1AK188R, had no effect on cTnT exon 5 splicing (Fig. 3E), indicating that the kinase activity of Dyrk1A is essential for its role in the regulation of cTnT splicing. SRp55 has three putative Dyrk1A sites, Ser-280, Ser-303, and Ser-316. To know which sites may mediate the role of Dyrk1A on SRp55-promoted cTnT exon 5 inclusion, we mutated these three sites to Alanine and determined their effects. We found that mutation of each sites decreased the cTnT exon 5 inclusion. Among the three sites, SRp55S280A had the strongest suppression effect in cTnT exon 5 inclusion (Fig. 3F). These results supported the finding that phosphorylation of SRp55 by Dyrk1A promotes cTnT exon 5 inclusion.
3.5. cTnT exon 5 inclusion is increased in the heart of Ts65Dn mice Alternative splicing of cTnT exon 5 is regulated developmentally. cTnT isoforms with exon 5 are expressed mainly in the pre- and neonatal stages, and gradually cTnT isoforms with exon 5 stop being expressed after birth [3,18]. To investigate the impact of Dyrk1A overexpression on cTnT exon 5 splicing in vivo, we determined the splicing products of cTnT exon 5 in the neonatal heart of Ts65Dn mice, a commonly used mouse model of DS in which Dyrk1A is overexpressed. As expected, we observed an approximate increase of 50% in the level of Dyrk1A in the heart tissue of Ts65Dn mice compared to their wild type littermates (Fig. 4A and 4B). The neonatal hearts expressed four isoforms of cTnT by alternative splicing of exons 4 and 5 [14]. Isoforms of cTnT with exon 5 were expressed in P5 and decreased during later development (Fig. 4C). Compared to the wild type littermates, the shift from cTnT exon 5 inclusion to exclusion was delayed obviously at around P10 in Ts65Dn hearts (Fig. 4C). To confirm this observation, we further determined cTnT exon 5 inclusion/exclusion in the hearts from Ts65Dn littermates at P10 by running PAGE gels to separated the cTnT isoform bands. We found that the ratio of cTnT E5þ/E5-in the
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Ts65Dn mice was ~1.5-fold higher than that of the control mice (Fig. 4D). Thus, overexpression of Dyrk1A promotes exon 5 inclusion in vivo and may cause a delay in exon 5 exclusion during neonatal cardiac development.
4. Discussion cTnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration. Transcripts for this gene undergo alternative splicing, which results in many tissue-specific isoforms [15]; however, the full-length nature of some of these variants has not yet been determined. Mutations to TNNT2 may be associated with mild or absent hypertrophy and predominant restrictive disease, with a high risk of sudden cardiac death [16]. Advancement to dilated cardiomyopathy may be more rapid in patients with TNNT2 mutations than in those with myosin heavy chain mutations [17,18]. No mutations have been shown to occur in the N- terminus of human cTnT. However, Biesiadecki et al. reported that cTnT N-terminal exons in several mammals undergo aberrant splicing resulting in DCM [19], indicating that abnormalities in cTnT mRNA splicing may play a role in the pathogenesis of congenital heart disease. The exclusion of exon 4 from adult human cTnT is responsible for the low molecular weight of TnT4 found in hypertrophic and failing hearts [2]. The alternative splicing of exon 5 in cTnT is tightly regulated during development such that it is normally excluded from all adult cTnT [3]. By analyzing Ts65Dn transgenic mouse hearts, we found that the expression of embryonic cTnT including exon 5 was increased (Fig. 4D). This was positively correlated with a slight reduction in Ts65Dn mouse heart weight (data not shown). These data support the notion that the overexpression of Dyrk1A in Ts65Dn mouse hearts may have a negative effect on cardiac development. Therefore, it may constitute a pathogenic factor in the development of congenital heart disease. Congenital heart disease occurs in 41%e56% of all DS patients
Fig. 4. Developmental switch off of cTnT exon 5 is delayed in Ts65Dn mice. A. Dyrk1A was overexpressed in the hearts from Ts65Dn mice. Homogenates from mouse hearts at P5, P10, P15 were analyzed by Western blots developed with anti-Dyrk1A and anti-b-actin. B. Quantifications of the blots after normalization with b-actin are presented as mean ± S.E.M. in the graph. C. Developmental switch off of cTnT exon 5 was delayed in Ts65Dn at P10. Total RNA of the mouse hearts from P5, P10, and P15 were extracted and then subjected to RT-PCR. D. The ratio of cTnT isoforms with and without exon 5 was increased in Ts65Dn. The alternative splicing of cTnT exon 5 in mouse hearts at P10 was measured byRT-PCR and separated by PAGE gel. The relative ratio of cTnT isoforms with exon 5 (E5þ) and without exon 5 (E5-) was calculated and the data were presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
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[20e23]. Analyses of the expression profiles of individual trisomic genes responsible for the DS phenotype have been performed in recent years [24,25]. Heart defects were correlated with the overexpression of 20 genes in mouse models of this human genetic disease [26]. Among these genes, Dyrk1A has been proposed as a causative gene for DS associated heart defects. Our group has reported that Dyrk1A is an important SR protein kinase and is involved in regulating tau pre-mRNA splicing [7e10]. Here, we found that its overexpression in mammalian cells and Ts65Dn mouse hearts promoted cTnT exon 5 splicing, providing evidence for its role in pre-mRNA alternative splicing. We previously found that Dyrk1A interacts with SRp55 through RRM domains and phosphorylates SRp55 mainly with RS1 and prorich domains [10]. In this study, the interaction of Dyrk1A with SRp55 was confirmed in cardiac cells and was shown to promote cTnT exon 5 inclusion. The role of Dyrk1A in SRp55-mediated cTnT exon 5 inclusion is SRp55 C-terminus dependent (Fig. 2D), suggesting that Dyrk1A may modulate SRp55 function by phosphorylating SRp55 at C terminus. By phosphorylation of SRp55 at Dyrk1A favorite site Ser-280, Dyrk1A enhanced SRp55's splicing activity and causes an increase in cTnT E5þ isoforms. In conclusion, we found that Dyrk1A regulates SRp55modulated cTnT exon 5 splicing. We identified the novel splicing events in Ts65Dn mice during neonatal cardiac development. These changes in cTnT exon 5 splicing may contribute to the pathogenesis of heart disorders in DS patients. Acknowledgments This work was supported in part by funds from Natural Science Foundation of Nantong University (14ZY020); National Natural Science Foundation of China Grant (grant 81503077); Basic Research Program of Jiangsu Education Department Grant (grant 13KJB320015); and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD); The Open Program of Key Laboratory Foundation of Capital Medical University (1300-1140170555).
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Transparency document [20]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.004
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