Inhibition of viral gene expression by human ribonuclease P

Kawa, Diane; Jun, Wang; Yuan, Yan; Liu, Fenyong

doi:10.1017/s1355838298980918

Cited by 62 publications

(135 citation statements)

References 31 publications

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“…Extensive studies have been carried out to develop strategies to express these ribozymes that colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (4,9,(21)(22)(23)(24). These studies have led to significant improvements of the efficacies of hammerhead and hairpin ribozymes in cellular environments.In our previous studies, M1GS RNA was targeted to a region of TK mRNA that is accessible to modification by dimethyl sulfate in cell culture and also accessible to ribozyme binding (19,25). Moreover, the ribozyme was expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA (19,23,25,26).…”

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confidence: 99%

“…Moreover, the ribozyme was expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA (19,23,25,26).…”

mentioning

confidence: 99%

“…In our previous studies, M1GS RNA was targeted to a region of TK mRNA that is accessible to modification by dimethyl sulfate in cell culture and also accessible to ribozyme binding (19,25). Moreover, the ribozyme was expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA (19,23,25,26).…”

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confidence: 99%

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RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

Kilani¹,

Trang

et al. 2000

Journal of Biological Chemistry

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An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G 224 G 225 3 AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k cat /K m ) than that derived from the wild type ribozyme. Our results suggest that the mutated A 224 A 225 are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (1-3). For example, both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, while a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (4 -9). Thus, ribozymes can be used as a tool in both basic and clinical research, such as in studies of tumorigenesis and antiviral gene therapy.RNase P is a ribonucleoprotein complex responsible for the 5Ј maturation of tRNAs (10, 11). It catalyzes a hydrolysis reaction to remove a 5Ј leader sequence from tRNA precursors (ptRNA) 1 and several other small RNAs . In Escherichia coli, RNase P consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (10, 11). In the presence of a high concentration of salt, such as 100 mM Mg 2ϩ , M1 RNA acts as a catalyst and cleaves ptRNAs in vitro in the absence of C5 protein (12). Extensive studies with both phylogenetic and biochemical analyses have established models for the secondary and threedimensional structures of RNase P catalytic RNAs (13-16). These models provide a framework to identify the putative active site and substrate binding site, and to study the mechanism of RNase P catalytic RNAs.Studies on substrate recognition by RNase P have revealed that a small model substrate can be cleaved efficiently by M1 ribozyme (Fig. 1A). This model substrate contains a structure equivalent to the acceptor stem, the T-stem, the 3Ј CCA sequence, and the 5Ј leader sequence of a ptRNA molecule. Accordingly, M1 catalytic RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence (external guide sequence) (Fig. 1A) (17). Moreover, a sequen...

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“…Moreover, the ribozyme was expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA (19,23,25,26).…”

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confidence: 99%

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RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

Kilani¹,

Trang

et al. 2000

Journal of Biological Chemistry

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151

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“…Reduction in both TK mRNA and protein levels was observed when EGSs were expressed in human cells infected with HSV-1 [81]. When EGS RNAs targeting HCMV mRNAs were expressed in human cells infected with HCMV, they were also effective in inducing RNase P to inhibit the expression of the target mRNAs and block HCMV growth [82].…”

Section: Antiviral Agentsmentioning

confidence: 99%

“…Thus, the EGS-based technology is highly specific and does not generate nonspecific "irrelevant cleavage" that is observed in RNase H-mediated cleavage induced by conventional antisense phosphorothioate molecules [36,38]. Third, EGSs exhibit little sign of cytotoxicity because cells expressing these molecules for more than 40 days appear to be normal [36,38,44,81].…”

Section: Advantage and Disadvantage Of M1gs And Rnase P-egs Technologymentioning

confidence: 99%

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

Kim

Liu

2007

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In E. coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications.

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A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1 1 1Edited by J. Doudna

Trang

Kilani

Kim

et al. 2000

Journal of Molecular Biology

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Inhibition of viral gene expression by human ribonuclease P

Cited by 62 publications

References 31 publications

RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1 1 1Edited by J. Doudna

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