Abstract
A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella A, Estivill X, Lazaro C (2003) Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 40:e82
Asparuhova MB, Marti G, Liu S, Serhan F, Trono D, Schümperli D (2007) Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J Gene Med 9:323–334
Ast G (2004) How did alternative splicing evolve? Nat Rev Genet 5:773–782
Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31:3568–3571
Gorlin RJ (1987) Nevoid basal-cell carcinoma syndrome. Medicine (Baltimore) 66:98–113
Gorman L, Suter D, Emerick V, Schümperli D, Kole R (1998) Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proc Natl Acad Sci USA 95:4929–4934
Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O (2004) Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306:1796–1799
Grimm C, Stefanovic B, Schümperli D (1993) The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J 12:1229–1238
Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G, Wainwright B, Bale AE (1996) Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85:841–851
Imai Y, Matsushima Y, Sugimura T, Terada M (1991) A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res 19:2785
Johnson JM, Castle J, Garrett-Engele P, Kan Z, Loerch PM, Armour CD, Santos R, Schadt EE, Stoughton R, Shoemaker DD (2003) Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302:2141–2144
Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein EH Jr, Scott MP (1996) Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272:1668–1671
Katsumata N, Ohtake M, Hojo T, Ogawa E, Hara T, Sato N, Tanaka T (2002) Compound heterozygous mutations in the cholesterol side-chain cleavage enzyme gene (CYP11A) cause congenital adrenal insufficiency in humans. J Clin Endocrinol Metab 87:3808–3813
Kralovicova J, Christensen MB, Vorechovsky I (2005) Biased exon/intron distribution of cryptic and de novo 3′ splice sites. Nucleic Acids Res 33:4882–4898
Krawczak M, Reiss J, Cooper DN (1992) The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 90:41–54
Madocsai C, Lim SR, Geib T, Lam BJ, Hertel KJ (2005) Correction of SMN2 Pre-mRNA splicing by antisense U7 small nuclear RNAs. Mol Ther 12:1013–1022
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66–71
Morohashi K, Sogawa K, Omura T, Fujii-Kuriyama Y (1987) Gene structure of human cytochrome P-450(SCC), cholesterol desmolase. J Biochem (Tokyo) 101:879–887
Nagao K, Togawa N, Fujii K, Uchikawa H, Kohno Y, Yamada M, Miyashita T (2005a) Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays. Hum Mol Genet 14:3379–3388
Nagao K, Toyoda M, Takeuchi-Inoue K, Fujii K, Yamada M, Miyashita T (2005b) Identification and characterization of multiple isoforms of a murine and human tumor suppressor, patched, having distinct first exons. Genomics 85:462–471
Nakai K, Sakamoto H (1994) Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene 141:171–177
Roca X, Sachidanandam R, Krainer AR (2003) Intrinsic differences between authentic and cryptic 5′ splice sites. Nucleic Acids Res 31:6321–6333
Scholl T, Pyne MT, Russo D, Ward BE (1999) BRCA1 IVS16 + 6T–>C is a deleterious mutation that creates an aberrant transcript by activating a cryptic splice donor site. Am J Med Genet 85:113–116
Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174
Sierakowska H, Sambade MJ, Agrawal S, Kole R (1996) Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides. Proc Natl Acad Sci USA 93:12840–12844
Teraoka SN, Telatar M, Becker-Catania S, Liang T, Onengut S, Tolun A, Chessa L, Sanal O, Bernatowska E, Gatti RA, Concannon P (1999) Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet 64:1617–1631
Vacek MM, Ma H, Gemignani F, Lacerra G, Kafri T, Kole R (2003) High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA. Blood 101:104–111
Acknowledgments
We are grateful to Dr. Daniel Schümperli for providing U7 SmOPT. We thank Mayu Yamazaki-Inoue for her technical support. This work was supported by the Naito Foundation and Grants for Cancer Research from the Ministry of Health, Labour and Welfare and a Grant-in-Aid for Scientific Research and the Budget for Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
10038_2007_192_MOESM1_ESM.doc
Supplementary Table List of primers used in this study. Restriction sites added to facilitate subcloning are underlined. (DOC 50 kb)
Rights and permissions
About this article
Cite this article
Uchikawa, H., Fujii, K., Kohno, Y. et al. U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites. J Hum Genet 52, 891–897 (2007). https://doi.org/10.1007/s10038-007-0192-8
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1007/s10038-007-0192-8
Keywords
This article is cited by
-
Nevoid basal cell carcinoma syndrome caused by splicing mutations in the PTCH1 gene
Familial Cancer (2017)
-
Lessons from non-canonical splicing
Nature Reviews Genetics (2016)
-
Novel synonymous substitution in POMGNT1 promotes exon skipping in a patient with congenital muscular dystrophy
Journal of Human Genetics (2008)
