5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing

S. Gary Mansfield, Rebecca Hawkins Clark, M. Puttaraju, Jolanta Kole, Jonathan A. Cohn, Lloyd G. Mitchell, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3′ exon replacement (3′ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5′ end of mRNA can be completely rewritten by 5′ER. For proof-of-concept, and to test whether 5′ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (ΔF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3′ splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5′ end of mRNA, and (2) efficiency can be improved substantially.

Original languageEnglish (US)
Pages (from-to)1290-1297
Number of pages8
JournalRNA
Volume9
Issue number10
DOIs
StatePublished - Oct 1 2003
Externally publishedYes

Fingerprint

Trans-Splicing
Spliceosomes
Exons
Introns
Messenger RNA
Anions
RNA Splice Sites
HEK293 Cells
Factor VIII
Knockout Mice
Epithelium
Epithelial Cells
RNA

Keywords

  • CFTR
  • Genetic disease
  • MRNA repair
  • SMaRT
  • Trans-splicing

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Mansfield, S. G., Clark, R. H., Puttaraju, M., Kole, J., Cohn, J. A., Mitchell, L. G., & Garcia-Blanco, M. (2003). 5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing. RNA, 9(10), 1290-1297. https://doi.org/10.1261/rna.5101903

5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing. / Mansfield, S. Gary; Clark, Rebecca Hawkins; Puttaraju, M.; Kole, Jolanta; Cohn, Jonathan A.; Mitchell, Lloyd G.; Garcia-Blanco, Mariano.

In: RNA, Vol. 9, No. 10, 01.10.2003, p. 1290-1297.

Research output: Contribution to journalArticle

Mansfield, SG, Clark, RH, Puttaraju, M, Kole, J, Cohn, JA, Mitchell, LG & Garcia-Blanco, M 2003, '5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing', RNA, vol. 9, no. 10, pp. 1290-1297. https://doi.org/10.1261/rna.5101903
Mansfield SG, Clark RH, Puttaraju M, Kole J, Cohn JA, Mitchell LG et al. 5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing. RNA. 2003 Oct 1;9(10):1290-1297. https://doi.org/10.1261/rna.5101903
Mansfield, S. Gary ; Clark, Rebecca Hawkins ; Puttaraju, M. ; Kole, Jolanta ; Cohn, Jonathan A. ; Mitchell, Lloyd G. ; Garcia-Blanco, Mariano. / 5′ Exon replacement and repair by spliceosome-mediated RNA trans-splicing. In: RNA. 2003 ; Vol. 9, No. 10. pp. 1290-1297.
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