Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2003 Aug;112(3):312-8.
doi: 10.1172/JCI19386.

Ribozyme-mediated revision of RNA and DNA

Meredith B Long  1 , J P Jones 3rd, Bruce A Sullenger, Jonghoe Byun
Affiliations
Review

Ribozyme-mediated revision of RNA and DNA

Meredith B Long et al. J Clin Invest. 2003 Aug.
No abstract available

PubMed Disclaimer

Figures

Figure 1
Figure 1
Comparison of self-splicing versus trans-splicing. The trans-splicing reaction (a) is similar to the natural self-splicing reaction (b) except that the target RNA is not covalently attached to the ribozyme. Exon sequences (blue and green) are shown as boxes and capital letters. Intron sequences (red) are shown as solid lines with lowercase letters. IGS, P1, and P10 helices are shown. The targeted uridine residue is shown as a bold letter and the splicing junction (S/J) is indicated. The exogenous guanosine nucleophile and terminal guanosine nucleotide of the ribozyme are shown as G (black) and g (red) respectively. A trans-splicing ribozyme binds a substrate RNA by forming bp’s (N-n) with the targeted transcript.
Figure 2
Figure 2
Schematic representation of RNA repair using a trans-splicing ribozyme. A Group I ribozyme delivers corrective sequences (3′ exon) to a mutant transcript. The ribozyme binds upstream of the mutation through base-pairing. Once bound, the ribozyme cleaves the target RNA, releases the downstream cleavage product, and splices the 3′ exon sequence onto the upstream cleavage product. M, mutant sequence; W, wild-type sequence corresponding to the mutated region.
Figure 3
Figure 3
Group II intron mobility and revision of DNA. Mechanism of Group II L1.LtrB intron mobility. (a) A Group II intron (red) is found in the ltrB gene (green) of Lactococcus lactis between exons E1 and E2. The intron encodes the LtrA gene product (blue). Following transcription of the ltrB gene, the LtrA protein is translated from the intron-containing precursor. The LtrA protein facilitates self-splicing of the L1.LtrB intron. The excised intron is in the form of a lariat that is bound to the LtrA protein in an RNP particle. The assembled RNP targets an intronless allele for insertion into the DNA. The RNA component reverse-splices into the sense strand at the junction between E1 and E2. The LtrA protein then cleaves the antisense strand downstream of the intron insertion site, providing a template for reverse transcription by the LtrA protein. Second-strand synthesis and DNA repair complete the intron’s mobilization into the target DNA. (b) Target-site recognition by the retrohoming RNP is shown in greater detail. The EBSs of the intron (EBS1, EBS2, and δ) pair with IBSs IBS1, IBS2, and δ′ in the target (shown in red). Two bp’s, at –21 and +5 (shown in blue), strongly influence the activity of the LtrA protein during retrohoming (6). Cleavage sites are shown by the arrows. The top (sense) strand is cleaved by the RNA. LtrA cleaves the bottom strand following reverse splicing of the intron.
Figure 4
Figure 4
Rational design and selection of retargeted introns using a mobility assay in E. coli. (a) Rational design. Putative target sites may be searched in a gene of interest using the consensus sequence shown (N is any nucleotide). At positions where a subset of nucleotides is preferred by the LtrA protein, each preferred nucleotide is listed (in blue). The EBS of the intron is designed to base pair with the predicted IBS of the target (in red). Mobility is assessed in E. coli using donor and recipient plasmids as shown in b. (b) Selection of target sites. Retrohoming into the recipient plasmid activates expression of the promoterless tetracycline resistance gene (tetR, yellow box). The donor plasmid expresses the intron (L1.LtrB, in red) and associated protein (LtrA, in blue) following transformation into E. coli and isopropyl-β-D-thiogalactopyranoside (IPTG) induction of the T7lac promoter (PT7lac). The intron is flanked by exon sequences (E1 and E2) required for self-splicing. **A library of introns is generated by randomizing EBS sequences in the intron. IBS sequences in E1 are also randomized to facilitate forward splicing of the library of precursors. A T7 promoter (yellow arrowhead) is located in domain IV of the intron. Selectable markers on donor and recipient plasmids are shown (ampR, camR). Transcription termination signals prevent TetR expression in the absence of retrohoming. Retargeted introns are recovered from ampR, tetR colonies.
See this image and copyright information in PMC

References

    1. Cech, T.R. 1993. Structure and mechanism of the large catalytic RNAs: Group I and group II introns and ribonuclease P. In The RNA World. R.F. Gesteland and J.F. Atkins, editors. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, USA. 239–269.
    1. Sullenger BA, Cech TR. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature. 1994;371:619–622. - PubMed
    1. Puttaraju M, Jamison SF, Mansfield SG, Garcia-Blanco MA, Mitchell LG. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat. Biotech. 1999;17:246–252. - PubMed
    1. Garcia-Blanco, M. 2003. Messenger RNA reprogramming by spliceosome mediated RNA trans-splicing. J. Clin. Invest. In press. - PMC - PubMed
    1. Guo H, et al. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science. 2000;289:452–457. - PubMed