pubmed.ncbi.nlm.nih.gov

A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs - PubMed

  • ️Sat Jan 01 2000

A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs

C E Reid et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Hepatitis delta virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5'OH and 2',3'-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5' hydroxyl and 2', 3'- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Processing pathways of precursor RNAs. Ribozymes are depicted as rectangles adjacent to their respective cleavage sites (circles). For HDV genomic ribozymes, recognition and cleavage of the sequence U/GGCCGGC occurs between the U and G residues. The downstream-cleaving hammerhead ribozyme cleaves after the sequence GUC in its substrate domain. L, M, and R correspond to the leftward, middle, and rightward cleavage fragments, respectively, generated by ribozyme cleavage. (A) Two wild-type HDV ribozymes. The open rectangle depicts attenuator sequences. MC represents circular (ligated) RNA. (B) Mutant (downstream-cleaving) and wild-type ribozymes. (C) Ribozyme-based intron.

Figure 2
Figure 2

Processing of HDV RNAs containing wild-type and mutant HDV ribozymes. (A) In vitro transcription. (B) Northern blot analysis of RNA harvested from transfected Huh7 cells. (C) Northern blot analysis of RNA generated from plasmid pDL669 harvested from Huh7 cells (lane 2) and from E. coli cells (lane 3). M, marker. Lane 1, HDV RNA generated from plasmid pDL625 (wild-type) and processed as in Fig. 1A. Product sizes (in nucleotides): LMR, 651; MR, 597; LM, 402; M, 348; R, 249. Lanes 2 and 3, HDV RNA generated from plasmid pDL669 (mutant) and processed as in Fig. 1B. Product sizes (in nucleotides): LMR, 756; MR, 638; LM, 484; M, 369. Percentages are percent polyacrylamide. The end-labeled DNA marker (M) is plasmid pDL616 linearized with MspI (7). Fragment lengths of the DNA marker are notated in A.

Figure 3
Figure 3

Processing of KanR RNA containing wild-type and mutant HDV ribozymes. (A) In vitro transcription. M on the left is plasmid pDL616 linearized with MspI. Marker on the right is ΦX174 linearized with HaeIII. (B) Northern blot analysis of RNA harvested from transfected Huh7 cells. Lane 1, HDV RNA generated from plasmid pDL669 and processed as in Fig. 1B. Lane 2, KanR RNA generated from plasmid pCR9 and processed as in Fig. 1B. Product sizes (in nucleotides): LMR, 1,217; MR, 1.112; LM, 968; M, 863. Lane 3, KanR RNA generated from plasmid pKW23 and processed as in Fig. 1B. Product sizes (in nucleotides): LMR, 1,228; MR, 1,112; LM, 979; M, 863. The mutant downstream-cleaving ribozyme in lane 3 contains clamp sequences that are missing from the corresponding ribozyme in 2. Lane 4, HDV RNA generated from plasmid pTW102 and processed as in Fig. 1A. Product sizes (in nucleotides): LMR, 1,468; MR, 1,414; LM, 1,219; M, 1,165. Percentages are percent polyacrylamide.

Figure 4
Figure 4

Processing of TetR RNA containing hammerhead and HDV genomic ribozymes. (A) In vitro transcription. (B) Northern blot analysis of RNA harvested from transfected Huh7 cells. M, marker. Lane 1, TetR RNA generated from plasmid pCR41 and processed as in Fig. 1B. Product sizes (nucleotides): LMR, 648; MR, 597; LM, 399; M, 348; R, 249. Percentages are percent polyacrylamide. DNA marker (M) sizes are defined in A.

Figure 5
Figure 5

Processing of CatR RNA containing an HDV ribozyme-based intron. Northern blot analysis of RNA harvested from Huh7 cells transfected with plasmid pCR36. Plasmid pCR36 generates CatR RNA, which is processed as in Fig. 1C. Product sizes (in nucleotides): LMR, 604; MR, 489; LR, 389; LM, 330; R, 274; M, 215; L, 115. Fragment-specific (L, M, R, etc.) 32P-labeled probes were used individually on independent blots of the RNA sample. M* is a darker exposure of M.

Similar articles

Cited by

References

    1. Rizzetto M, Hoyer B, Canese M G, Shih J W, Purcell R H, Gerin J L. Proc Natl Acad Sci USA. 1980;77:6124–6128. - PMC - PubMed
    1. Kuo M Y, Goldberg J, Coates L, Mason W, Gerin J, Taylor J. J Virol. 1988;62:1855–1861. - PMC - PubMed
    1. Zuker M, Stiegler P. Nucleic Acids Res. 1981;9:133–148. - PMC - PubMed
    1. Branch A D, Robertson H D. Science. 1984;223:450–455. - PubMed
    1. Wadkins T S, Perrotta A T, Ferre-D'Amare A R, Doudna J A, Been M D. RNA. 1999;5:720–727. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources