Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia
- Mayumi Yoshidaa,
- Naoyuki Kataokab,1,
- Kenjyo Miyauchic,
- Kenji Ohea,d,
- Kei Iidaa,e,
- Suguru Yoshidaf,
- Takayuki Nojimag,
- Yukiko Okunoa,e,
- Hiroshi Onogia,h,
- Tomomi Usuii,
- Akihide Takeuchia,
- Takamitsu Hosoyaf,
- Tsutomu Suzukic, and
- Masatoshi Hagiwaraa,1
- aDepartment of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan;
- bLaboratory for Malignancy Control Research, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan;
- cDepartment of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-8510, Japan;
- dKyoto University Graduate School of Medicine, Kyoto 606-8501, Japan;
- eMedical Research Support Center, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan;
- fLaboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan;
- gSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;
- hKinoPharma, Inc., Tokyo 154-0024, Japan; and
- iLaboratory of Gene Expression, School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
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Edited by Gideon Dreyfuss, University of Pennsylvania, Philadelphia, PA, and approved December 30, 2014 (received for review August 12, 2014)
Significance
Familial dysautonomia (FD) is caused by missplicing of the IκB kinase complex-associated protein (IKAP) gene, which results in the skipping of exon 20, especially in neurons. FD would be treatable if exon 20 inclusion were increased correctly to reestablish correct splicing. Here, we have established a dual-color splicing reporter that recapitulates FD-type splicing. By using this reporter, we have identified a small chemical compound, named rectifier of aberrant splicing (RECTAS), that rectifies the aberrant splicing of FD. RECTAS promotes both exon 20 inclusion and the product IKAP expression in cells of patients with FD. Furthermore, we have demonstrated that modification levels of wobble uridine residues of several tRNAs are reduced in FD cells and that RECTAS can recover not only tRNA modifications but also cell viability of FD cells.
Abstract
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.
Footnotes
- ↵1To whom correspondence may be addressed. Email: kataoka.naoyuki.6m{at}kyoto-u.ac.jp or hagiwara.masatoshi.8c{at}kyoto-u.ac.jp.
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Author contributions: M.Y., N.K., T.N., T.H., T.S., and M.H. designed research; M.Y., N.K., K.M., K.O., K.I., S.Y., H.O., and T.U. performed research; M.Y., N.K., K.M., K.O., K.I., S.Y., T.N., Y.O., H.O., A.T., T.H., and T.S. contributed new reagents/analytic tools; M.Y., N.K., K.M., K.O., K.I., S.Y., T.H., T.S., and M.H. analyzed data; and M.Y., N.K., K.O., K.I., T.H., T.S., and M.H. wrote the paper.
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Conflict of interest statement: S.Y., T.H., and M.H. are coinventors on pending patent applications based on this work (Japanese patent application no. 2013-146891).
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This article is a PNAS Direct Submission.
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Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo [accession no. GSE58038 (transcriptome analysis of the effect of RECTAS on fibroblast cells derived from a familial dysautonomia patient)].
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See Commentary on page 2637.
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This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1415525112/-/DCSupplemental.
