RBM24 promotes U1 snRNP recognition of the mutated 5′ splice site in the<i>IKBKAP</i>gene of familial dysautonomia

Ohe, Kenji; Yoshida, Mayumi; Nakano-Kobayashi, Akiko; Hosokawa, Motoyasu; Sako, Y; Sakuma, Masayuki; Okuno, Yukiko; Usui, Tomomi; Ninomiya, K.; Nojima, Takayuki; Kataoka, Naoyuki; Hagiwara, Masatoshi

doi:10.1261/rna.059428.116

Cited by 14 publications

(21 citation statements)

References 52 publications

(71 reference statements)

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“…Consistent with our observation, Ohe et al . recently identified an inhibitory element in this region ( 31 ). The enhancement of IKBKAP exon 20 splicing was reduced as the ASOs targeted regions farther away from the 5′ splice site.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Ohe et al . showed that the IVS20 +13 ∼ +29 region, which overlaps with our ASO target site, contains a splicing silencer element that enhances IKBKAP exon 20 splicing when deleted ( 31 ). Interestingly, this region also contains a binding site for RBM24, which enhances IKBKAP exon 20 inclusions in muscle cells and other tissues with high levels of RBM24 expression ( 31 ).…”

Section: Discussionmentioning

confidence: 99%

“…showed that the IVS20 +13 ∼ +29 region, which overlaps with our ASO target site, contains a splicing silencer element that enhances IKBKAP exon 20 splicing when deleted ( 31 ). Interestingly, this region also contains a binding site for RBM24, which enhances IKBKAP exon 20 inclusions in muscle cells and other tissues with high levels of RBM24 expression ( 31 ). This finding suggests that the tissue-specific IKBKAP exon 20 splicing defect caused by the major FD mutation may be affected by differential expression and binding of splicing regulators to intron 20.…”

Section: Discussionmentioning

confidence: 99%

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Antisense oligonucleotides correct the familial dysautonomia splicing defect in IKBKAP transgenic mice

Sinha

Kim

Nomakuchi

et al. 2018

Nucleic Acids Research

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Familial dysautonomia (FD) is a rare inherited neurodegenerative disorder caused by a point mutation in the IKBKAP gene that results in defective splicing of its pre-mRNA. The mutation weakens the 5′ splice site of exon 20, causing this exon to be skipped, thereby introducing a premature termination codon. Though detailed FD pathogenesis mechanisms are not yet clear, correcting the splicing defect in the relevant tissue(s), thus restoring normal expression levels of the full-length IKAP protein, could be therapeutic. Splice-switching antisense oligonucleotides (ASOs) can be effective targeted therapeutics for neurodegenerative diseases, such as nusinersen (Spinraza), an approved drug for spinal muscular atrophy. Using a two-step screen with ASOs targeting IKBKAP exon 20 or the adjoining intronic regions, we identified a lead ASO that fully restored exon 20 splicing in FD patient fibroblasts. We also characterized the corresponding cis-acting regulatory sequences that control exon 20 splicing. When administered into a transgenic FD mouse model, the lead ASO promoted expression of full-length human IKBKAP mRNA and IKAP protein levels in several tissues tested, including the central nervous system. These findings provide insights into the mechanisms of IKBKAP exon 20 recognition, and pre-clinical proof of concept for an ASO-based targeted therapy for FD.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Antisense oligonucleotides correct the familial dysautonomia splicing defect in IKBKAP transgenic mice

Sinha

Kim

Nomakuchi

et al. 2018

Nucleic Acids Research

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“…Exonic and intronic sequences are in upper and lower case, respectively. The recently described intronic splicing enhancer ( 48 ) is indicated. ( B ) Co-transfection experiment of ELP1 minigenes with ExSpeU1s (Ik10–72) and modified-U7 snRNA (U7-Ik10) in Hek293T cells.…”

Section: Resultsmentioning

confidence: 99%

Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model

Donadon

Pinotti

Rajkowska

et al. 2018

Human Molecular Genetics

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Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204+6T>C) that negatively affects the definition of exon 20 in the elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 5′ splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, exon-specific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1s-adeno-associated virus particles as a novel therapeutic strategy for FD.

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“…Several potential therapies for FD have been investigated including PS [30][31][32]34 , kinetin 40,41 , tocotrienols 47,48 , and the green tea component epigallocatechin gallate 49 . Other therapy strategies are based on the FD mutation acting by altering gene splicing in the nerve system in a tissue-specific manner 25,27,[50][51][52] . We focused on PS since it is a well-studied, FDA-approved food supplement that upregulates IKAP production and has no known side effects; therefore, we sought to identify other compounds that could be used in combination with PS to benefit patients.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial Treatment Increases IKAP Levels in Human Cells Generated from Familial Dysautonomia Patients

Ast

Yanai

Zonszain

et al. 2019

Preprint

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Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from a point mutation at the 5' splice site of intron 20 in the IKBKAP gene. This mutation decreases production of the IKAP protein, and treatments that increase the level of the full-length IKBKAP transcript are likely to be of therapeutic value. We previously found that phosphatidylserine (PS), an FDA-approved food supplement, elevates IKAP levels in cells generated from FD patients. Here we demonstrate that combined treatment of cells generated from FD patients with PS and kinetin or PS and the histone deacetylase inhibitor trichostatin A (TSA) resulted in an additive elevation of IKAP compared to each drug alone. This indicates that the compounds influence different pathways. We also found that pridopidine enhances production of IKAP in cells generated from FD patients. Pridopidine has an additive effect on IKAP levels when used in combination with kinetin or TSA, but not with PS; suggesting that PS and pridopidine influence IKBKAP levels through the same mechanism. Indeed, we demonstrate that the effect of PS and pridopidine is through sigma-1 receptor-mediated activation of the BDNF signaling pathway. A combination treatment with any of these drugs with different mechanisms has potential to benefit FD patients.

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RBM24 promotes U1 snRNP recognition of the mutated 5′ splice site in theIKBKAPgene of familial dysautonomia

Cited by 14 publications

References 52 publications

Antisense oligonucleotides correct the familial dysautonomia splicing defect in IKBKAP transgenic mice

Antisense oligonucleotides correct the familial dysautonomia splicing defect in IKBKAP transgenic mice

Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model

Combinatorial Treatment Increases IKAP Levels in Human Cells Generated from Familial Dysautonomia Patients

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