Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

Nishida, Atsushi; Kataoka, Naoyuki; Takeshima, Yasuhiro; Yagi, Minoru; Awano, Hiroyuki; M, Ota; Itoh, Kyoko; Hagiwara, Masatoshi; Matsuo, Masafumi

doi:10.1038/ncomms1306

Cited by 83 publications

(92 citation statements)

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“…Owing to the specific role of TG003 in inhibiting Clk family involved in SR protein phosphorylation, 12,13 we investigated whether TG003 modulates alternative splicing of TP53 intron 9. Indeed, no significant variation in the global expression of TAp53 mRNA variants (p53a, p53b and p53g) generated by the proximal promoter was observed in response to TG003 treatment, suggesting that TG003 increased p53b and p53g transcripts through a mechanism not affecting TP53 promoter activity (Supplementary Figure 4b).…”

Section: Resultsmentioning

confidence: 99%

“…12 In particular, TG003 inhibits Clk1/4 activity, resulting in SFRS1 dephosphorylation, which induced SFRS1 subcellular re-localization and inhibited the SFRS1-dependent splicing of globin mRNA. Moreover, TG003 has been shown to modulate alternative splicing in vitro and in vivo, 12,13 and to activate p53 inducing a p53-mediated cell response. 14 Here we investigated the effects of TG003 and SFRS1 on splicing of TP53 intron 9 and characterized the biological consequences.…”

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confidence: 99%

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Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

et al. 2014

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In addition to the tumor suppressor p53 protein, also termed p53a, the TP53 gene produces p53b and p53c through alternative splicing of exons 9b and 9c located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53b and p53c at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9b/9c. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and a variant, supporting our experimental data. Using siRNA specifically targeting exons 9b/9c, we demonstrate that cell growth can be driven by modulating p53b and p53c expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53b and p53c promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53b enhanced p53a transcriptional activity on the p21 and Bax promoters, while p53c increased p53a transcriptional activity on the Bax promoter only. Moreover, p53b and p53c co-immunoprecipitate with p53a only in the presence of p53-responsive promoter. Interestingly, although p53b and p53c promote apoptosis in MCF7 cells, p53b and p53c maintain cell growth in response to TG003 in a p53a-dependent manner. The dual activities of p53b and p53c isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53b and p53c regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. including C-terminal isoforms produced by alternative splicing of intron 9 3 (Figure 1a). Exclusion of the entire intron 9 generates the canonical full-length p53 protein (or p53a), a transcription factor activated in response to diverse intracellular and extracellular signals (Figure 1b). Hence, p53 regulates gene expression to modulate cell-fate outcome by regulating biological processes, including apoptosis and cell cycle progression.5 Inclusion of alternative exons 9b and 9g contained in intron 9 gives rise to p53b and p53g protein isoforms that present new residues in place of the usual oligomerization domain present in p53a. Early studies reported that p53b and p53g retain characteristics of a tumor suppressor. 4 Indeed, p53b modulates p53a transcriptional activity in a promoter-dependent manner in response to stress 3 and promotes apoptosis and senescence, indicating that p53b can modulate p53a suppressive activity. 3,6 The biological significance of p53b and p53g isoforms is emphasized by clinical data. Both p53b and p53g expression is lost in B60% of breast cancer tumors.3,7 Furthermore, breast cancer patients expressing both mutant p53 and p53g ha...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

et al. 2014

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“…S3B), suggesting that RECTAS has therapeutic potential in FD. Artificial splicing rectification with RECTAS may offer an unexplored direction for pharmacological intervention in other hereditary diseases, such as Duchenne muscular dystrophy (30).…”

Section: Discussionmentioning

confidence: 99%

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia

Yoshida

Kataoka

Miyauchi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD. IKAP is currently known as elongator protein 1 (ELP1), an integral component of the human Elongator complex, which was originally identified in Saccharomyces cerevisiae and shown to be well conserved among species (1). Although multiple functions of IKAP/ELP1 in JNK signaling, neuronal development during embryogenesis, exocytosis, and actin cytoskeleton regulation have been reported (reviewed in refs. 2, 3), yeast genetic analyses have shown that the Elongator complex is also required for the formation of the C5-substituent of 5-carbamoylmethyl (ncm 5 ), 5-methoxycarbonylmethyl (mcm 5 ), and its derivatives at the wobble uridine in tRNAs recognizing purine-ending codons (4, 5). Most recently, it was demonstrated that conditional IKAP/Elp1 KO in mouse testes results in male infertility by disrupting meiotic progression, along with the reduction of modified nucleosides [5-methoxycarbonylmethyl uridine (mcm 5 U), 5-carbamoylmethyl uridine (ncm 5 U), and 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U)] of total tRNAs in the testes (6). These modifications are highly likely to play critical roles in the maintenance of translational fidelity, suggesting that the defects in these modifications lead to the mistranslation of various proteins.Familial dysautonomia (FD; Riley-Day syndrome), an autosomal recessive neurodegenerative disease, is characterized by impaired development and progressive degeneration of the sensory and autonomic nerves. Patients who have FD exhibit various symptoms, including cardiovascular instability, recurrent pneumonia, vomiting/dysautonomic crisis, gastrointestinal dysfunction, decreased sensitivity to pain and temperature, and defective lacrimation. FD is a very common disorder in the Ashkenazi Jewish population, with a carrier frequency of 1 in 27. More than 99% of patients who have FD harbor a homozygous mutation in intron 20 (IVS20 + 6T > C: FD mutation)...

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“…Candidate chemicals (kinetin, kinetin riboside, TG003, SRPIN340, RES, epigallocatechin gallate, 3-nitropropionic acid, and prednisolone were chosen based on their published splicing modulation activity [12][13][14][15][16]. SRT1720 (Calbiochem, San Diego, CA) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (Wako Pure Chemical Industries, Kyoto, Japan) were used at 0.1 lM and 1 mM, respectively.…”

Section: Chemicalsmentioning

confidence: 99%

Resveratrol enhances splicing of insulin receptor exon 11 in myotonic dystrophy type 1 fibroblasts

Takarada

Nishida

Takeuchi

et al. 2015

Brain and Development

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Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

Cited by 83 publications

References 53 publications

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia

Resveratrol enhances splicing of insulin receptor exon 11 in myotonic dystrophy type 1 fibroblasts

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