Use of A U16 snoRNA-containing Ribozyme Library to Identify Ribozyme Targets in HIV-1

Division of Molecular Biology, Beckman Research Institute of The City of Hope, Duarte, California 91010, USA.
Molecular Therapy (Impact Factor: 6.23). 07/2008; 16(6):1113-9. DOI: 10.1038/mt.2008.54
Source: PubMed


Hammerhead ribozymes have been shown to silence human immunodeficiency virus-1 (HIV-1) gene expression by site-specific cleavage of viral mRNA. The two major factors that determine whether ribozymes will be effective for post-transcriptional gene silencing are colocalization of the ribozyme and the target RNAs, and the choice of an appropriate target site on the mRNA. An effective screening strategy for potential targets on the viral genome is the use of ribozyme libraries in cell culture. Capitalizing on previous findings that HIV-1 and ribozymes can be colocalized in the nucleolus, we created a novel hammerhead ribozyme library by inserting hammerhead ribozymes with fully randomized stems 1 and 2 into the body of the U16 small nucleolar RNA (snoRNA). Following three rounds of cotransfection with an HIV-1 proviral DNA harboring the herpes simplex virus thymidine kinase (HSV-TK) gene, we selected for gancyclovir-resistant cells and identified a ribozyme sequence that could potentially target both the U5 and gag genes of HIV-1 regions on the HIV-1 genome through partial homologies with these targets. When the ribozymes were converted to full complementarity with the targets, they provided potent inhibition of HIV-1 replication in cell culture. These results provide a novel approach for identifying ribozyme targets in HIV-1.

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Available from: John J Rossi, Jun 22, 2015
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    • "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. "
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    ABSTRACT: 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.
    Molecular Therapy 07/2014; 3:e178. DOI:10.1038/mtna.2014.31 · 6.23 Impact Factor
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    • "The evolution of a ribozyme population in cells presented here and in a related study [10] is different from previous selection studies [46], [47], [48], [49], [50], because evolution introduces sequence diversity over multiple selection steps, whereas selection introduces sequence diversity only in the initial library [2]. Five previous studies selected ribozymes from ribozyme libraries in cells. "
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    ABSTRACT: How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.
    PLoS ONE 01/2014; 9(1):e86473. DOI:10.1371/journal.pone.0086473 · 3.23 Impact Factor
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    • "Randomization of these 7–9 nucleotides in each arm yields a large variety of ribozyme capable of targeting multiple mRNA substrates [11]. Such pool of degenerate ribozymes expressed from an exogenous promoter has been used as a tool for identification of genes involved in apoptosis, migration, invasion, differentiation and diseases [15]–[20]. In order to identify the cellular targets involved in cancer cell cytotoxity of Ashwagandha leaf extract (i-Extract), we infected the MCF7 cells with retrovirus driven randomized ribozyme library prior to the treatment. "
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    ABSTRACT: Ashwagandha is a popular Ayurvedic herb used in Indian traditional home medicine. It has been assigned a variety of health-promoting effects of which the mechanisms remain unknown. We previously reported the selective killing of cancer cells by leaf extract of Ashwagandha (i-Extract) and its purified component Withanone. In the present study, we investigated its mechanism by loss-of-function screening (abrogation of i-Extract induced cancer cell killing) of the cellular targets and gene pathways. Randomized ribozyme library was introduced into cancer cells prior to the treatment with i-Extract. Ribozymes were recovered from cells that survived the i-Extract treatment. Gene targets of the selected ribozymes (as predicted by database search) were analyzed by bioinformatics and pathway analyses. The targets were validated for their role in i-Extract induced selective killing of cancer cells by biochemical and molecular assays. Fifteen gene-targets were identified and were investigated for their role in specific cancer cell killing activity of i-Extract and its two major components (Withaferin A and Withanone) by undertaking the shRNA-mediated gene silencing approach. Bioinformatics on the selected gene-targets revealed the involvement of p53, apoptosis and insulin/IGF signaling pathways linked to the ROS signaling. We examined the involvement of ROS-signaling components (ROS levels, DNA damage, mitochondrial structure and membrane potential) and demonstrate that the selective killing of cancer cells is mediated by induction of oxidative stress. Ashwagandha leaf extract and Withanone cause selective killing of cancer cells by induction of ROS-signaling and hence are potential reagents that could be recruited for ROS-mediated cancer chemotherapy.
    PLoS ONE 10/2010; 5(10):e13536. DOI:10.1371/journal.pone.0013536 · 3.23 Impact Factor
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