Enhanced Expression and HIV-1 Inhibition of Chimeric tRNALys3-Ribozymes under Dual U6 snRNA and tRNA Promoters

Chang, Zongli; Westaway, Shawn K.; Li, Shirley Xin; Zaia, John A.; Rossi, John J.; Scherer, Lisa

doi:10.1006/mthe.2002.0696

Cited by 14 publications

(7 citation statements)

References 26 publications

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“…Indeed, a tRNA promoter is located within the tRNA scaffold (Chang, et al, 2002; Schramm and Hernandez, 2002). To test this, we expressed tBroccoli without using the U6 promoter.…”

Section: Resultsmentioning

confidence: 99%

In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

Filonov

Kam

Song

et al. 2015

Chemistry & Biology

159

200

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SUMMARY RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells, and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression.

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“…Indeed, a tRNA promoter is located within the tRNA scaffold (Chang, et al, 2002; Schramm and Hernandez, 2002). To test this, we expressed tBroccoli without using the U6 promoter.…”

Section: Resultsmentioning

confidence: 99%

In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

Filonov

Kam

Song

et al. 2015

Chemistry & Biology

159

200

View full text Add to dashboard Cite

show abstract

“…This behavior is not surprising since the mutated ribozyme can still bind to its target mRNA, and thus may elicit antisense-like inhibitory effects on protein production. In addition, it is conceivable that the short doublestranded regions in the mutant ribozyme±target RNA complex may attract intracellular double-strand-speci®c RNase activity, resulting in decreased target mRNAs as observed here for some clones (27). Also, small interfering RNA (siRNA)-like effects may potentially contribute to such decreases, although the strict sequence and length requirements for siRNAs make this less likely (28,29).…”

Section: Discussionmentioning

confidence: 96%

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

Clark¹

2003

Nucleic Acids Research

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In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.

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“…37 While no toxicity has been observed in this study, the moderate effect reported emphasizes the need to find more inhibitory molecules for use in gene therapy. Approaches that have been described to identify new Rz candidates include the use of (i) RNA Polymerase III (Pol III) promoters to achieve higher levels of expression, 38,39 (ii) cellular screens to identify optimal target sites, 18,19 (iii) chimeric Rzs to enhance anti-viral effects, 18,27,40,41 and (iv) alternative motifs such as modified RNase P and HDV Rzs. 7,16 In this study, we screened SOFA-HDV-Rzs, expressed from the RNA Pol III H1 promoter, targeting the 5′UTR and Gag coding sequence of HIV-1 RNA for effects on viral production.…”

Section: Discussionmentioning

confidence: 99%

A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

Scarborough

Lévesque

Boudrias-Dalle

et al. 2014

Molecular Therapy - Nucleic Acids

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Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

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Enhanced Expression and HIV-1 Inhibition of Chimeric tRNALys3-Ribozymes under Dual U6 snRNA and tRNA Promoters

Cited by 14 publications

References 26 publications

In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

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