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Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.
J Clin Invest. 1995 Feb; 95(2):515-20.JCI

Abstract

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy.

Authors+Show Affiliations

Division of Genetics, Kobe University School of Medicine, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7860733

Citation

Takeshima, Y, et al. "Modulation of in Vitro Splicing of the Upstream Intron By Modifying an Intra-exon Sequence Which Is Deleted From the Dystrophin Gene in Dystrophin Kobe." The Journal of Clinical Investigation, vol. 95, no. 2, 1995, pp. 515-20.
Takeshima Y, Nishio H, Sakamoto H, et al. Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J Clin Invest. 1995;95(2):515-20.
Takeshima, Y., Nishio, H., Sakamoto, H., Nakamura, H., & Matsuo, M. (1995). Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. The Journal of Clinical Investigation, 95(2), 515-20.
Takeshima Y, et al. Modulation of in Vitro Splicing of the Upstream Intron By Modifying an Intra-exon Sequence Which Is Deleted From the Dystrophin Gene in Dystrophin Kobe. J Clin Invest. 1995;95(2):515-20. PubMed PMID: 7860733.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. AU - Takeshima,Y, AU - Nishio,H, AU - Sakamoto,H, AU - Nakamura,H, AU - Matsuo,M, PY - 1995/2/1/pubmed PY - 1995/2/1/medline PY - 1995/2/1/entrez SP - 515 EP - 20 JF - The Journal of clinical investigation JO - J Clin Invest VL - 95 IS - 2 N2 - Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy. SN - 0021-9738 UR - https://www.unboundmedicine.com/medline/citation/7860733/Modulation_of_in_vitro_splicing_of_the_upstream_intron_by_modifying_an_intra_exon_sequence_which_is_deleted_from_the_dystrophin_gene_in_dystrophin_Kobe_ L2 - https://doi.org/10.1172/JCI117693 DB - PRIME DP - Unbound Medicine ER -