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Smith A, Zhang I, Trang P, Liu F. Engineering of RNase P Ribozymes for Therapy against Human Cytomegalovirus Infection. Viruses 2024; 16:1196. [PMID: 39205170 PMCID: PMC11360822 DOI: 10.3390/v16081196] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 07/20/2024] [Accepted: 07/22/2024] [Indexed: 09/04/2024] Open
Abstract
Nucleic acid-based gene interference and editing strategies, such as antisense oligonucleotides, ribozymes, RNA interference (RNAi), and CRISPR/Cas9 coupled with guide RNAs, are exciting research tools and show great promise for clinical applications in treating various illnesses. RNase P ribozymes have been engineered for therapeutic applications against human viruses such as human cytomegalovirus (HCMV). M1 ribozyme, the catalytic RNA subunit of RNase P from Escherichia coli, can be converted into a sequence-specific endonuclease, M1GS ribozyme, which is capable of hydrolyzing an mRNA target base-pairing with the guide sequence. M1GS RNAs have been shown to hydrolyze essential HCMV mRNAs and block viral progeny production in virus-infected cell cultures. Furthermore, RNase P ribozyme variants with enhanced hydrolyzing activity can be generated by employing in vitro selection procedures and exhibit better ability in suppressing HCMV gene expression and replication in cultured cells. Additional studies have also examined the antiviral activity of RNase P ribozymes in mice in vivo. Using cytomegalovirus infection as an example, this review summarizes the principles underlying RNase P ribozyme-mediated gene inactivation, presents recent progress in engineering RNase P ribozymes for applications in vitro and in mice, and discusses the prospects of using M1GS technology for therapeutic applications against HCMV as well as other pathogenic viruses.
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Affiliation(s)
- Adam Smith
- Program in Comparative Biochemistry, University of California, Berkeley, CA 94720, USA
- School of Public Health, University of California, Berkeley, CA 94720, USA
| | - Isadora Zhang
- School of Public Health, University of California, Berkeley, CA 94720, USA
| | - Phong Trang
- School of Public Health, University of California, Berkeley, CA 94720, USA
| | - Fenyong Liu
- Program in Comparative Biochemistry, University of California, Berkeley, CA 94720, USA
- School of Public Health, University of California, Berkeley, CA 94720, USA
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2
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Sridhara S. Multiple structural flavors of RNase P in precursor tRNA processing. WILEY INTERDISCIPLINARY REVIEWS. RNA 2024; 15:e1835. [PMID: 38479802 DOI: 10.1002/wrna.1835] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 01/26/2024] [Accepted: 01/29/2024] [Indexed: 06/06/2024]
Abstract
The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
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Affiliation(s)
- Sagar Sridhara
- Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden
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3
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Simmons TR, Ellington AD, Contreras LM. RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR. Methods Mol Biol 2022; 2518:1-31. [PMID: 35666436 DOI: 10.1007/978-1-0716-2421-0_1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.
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Affiliation(s)
- Trevor R Simmons
- McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA
| | - Andrew D Ellington
- Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA
| | - Lydia M Contreras
- McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA.
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4
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Pifer R, Greenberg DE. Antisense antibacterial compounds. Transl Res 2020; 223:89-106. [PMID: 32522669 DOI: 10.1016/j.trsl.2020.06.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 05/28/2020] [Accepted: 06/01/2020] [Indexed: 02/08/2023]
Abstract
Extensive antibiotic use combined with poor historical drug stewardship practices have created a medical crisis in which once treatable bacterial infections are now increasingly unmanageable. To combat this, new antibiotics will need to be developed and safeguarded. An emerging class of antibiotics based upon nuclease-stable antisense technologies has proven valuable in preclinical testing against a variety of bacterial pathogens. This review describes the current state of development of antisense-based antibiotics, the mechanisms thus far employed by these compounds, and possible future avenues of research.
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Affiliation(s)
- Reed Pifer
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas
| | - David E Greenberg
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas.
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5
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Jani S, Jackson A, Davies-Sala C, Chiem K, Soler-Bistué A, Zorreguieta A, Tolmasky ME. Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules. Methods Mol Biol 2018; 1737:89-98. [PMID: 29484589 DOI: 10.1007/978-1-4939-7634-8_6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.
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Affiliation(s)
- Saumya Jani
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA
| | - Alexis Jackson
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina
| | - Carol Davies-Sala
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina
| | - Kevin Chiem
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA
| | - Alfonso Soler-Bistué
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina
| | - Angeles Zorreguieta
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina
| | - Marcelo E Tolmasky
- Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA.
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Jackson A, Jani S, Sala CD, Soler-Bistué AJC, Zorreguieta A, Tolmasky ME. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biol Methods Protoc 2016; 1. [PMID: 27857983 PMCID: PMC5108630 DOI: 10.1093/biomethods/bpw001] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
External guide sequences (EGSs) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNAs) – modified ribonucleotides that contain a bridge at the 2ʹ,4ʹ-position of the ribose. LNA and the second-generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6ʹ)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5ʹ- and 3ʹ-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell-penetrating peptide, the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and biological activity.
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Affiliation(s)
- Alexis Jackson
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA
| | - Saumya Jani
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA
| | - Carol Davies Sala
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA; Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina
| | - Alfonso J C Soler-Bistué
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA; Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina
| | - Angeles Zorreguieta
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina
| | - Marcelo E Tolmasky
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA
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7
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RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences. Biomolecules 2015; 5:3029-50. [PMID: 26569326 PMCID: PMC4693268 DOI: 10.3390/biom5043029] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2015] [Revised: 10/16/2015] [Accepted: 10/29/2015] [Indexed: 01/06/2023] Open
Abstract
The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.
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Sala CD, Soler-Bistué A, Bonomo R, Zorreguieta A, Tolmasky ME. External guide sequence technology: a path to development of novel antimicrobial therapeutics. Ann N Y Acad Sci 2015; 1354:98-110. [PMID: 25866265 PMCID: PMC4600001 DOI: 10.1111/nyas.12755] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2014] [Revised: 02/14/2015] [Accepted: 03/03/2015] [Indexed: 12/11/2022]
Abstract
RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.
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Affiliation(s)
- Carol Davies Sala
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of
Buenos Aires, Argentina
- Center for Applied Biotechnology Studies, College of Natural Sciences and
Mathematics, California State University Fullerton, Fullerton, California
| | - Alfonso Soler-Bistué
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of
Buenos Aires, Argentina
- Center for Applied Biotechnology Studies, College of Natural Sciences and
Mathematics, California State University Fullerton, Fullerton, California
| | - Robert Bonomo
- Department of Medicine, Case Western Reserve University School of Medicine,
Cleveland, Ohio
| | - Angeles Zorreguieta
- Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of
Buenos Aires, Argentina
| | - Marcelo E. Tolmasky
- Center for Applied Biotechnology Studies, College of Natural Sciences and
Mathematics, California State University Fullerton, Fullerton, California
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9
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Dinan AM, Loftus BJ. (Non-)translational medicine: targeting bacterial RNA. Front Genet 2013; 4:230. [PMID: 24265632 PMCID: PMC3821060 DOI: 10.3389/fgene.2013.00230] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2013] [Accepted: 10/18/2013] [Indexed: 11/26/2022] Open
Abstract
The rise and spread of antibiotic resistance is among the most severe challenges facing modern medicine. Despite this fact, attempts to develop novel classes of antibiotic have been largely unsuccessful. The traditional mechanisms by which antibiotics work are subject to relatively rapid bacterial resistance via mutation, and hence have a limited period of efficacy. One promising strategy to ameliorate this problem is to shift from the use of chemical compounds targeting protein structures and processes to a new era of RNA-based therapeutics. RNA-mediated regulation (riboregulation) has evolved naturally in bacteria and is therefore a highly efficient means by which gene expression can be manipulated. Here, we describe recent advances toward the development of effective anti-bacterial therapies, which operate through various strategies centered on RNA.
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Affiliation(s)
- Adam M Dinan
- School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin Dublin, Ireland
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10
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Sawyer AJ, Wesolowski D, Gandotra N, Stojadinovic A, Izadjoo M, Altman S, Kyriakides TR. A peptide-morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model. Int J Pharm 2013; 453:651-5. [PMID: 23727592 DOI: 10.1016/j.ijpharm.2013.05.041] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2013] [Revised: 04/24/2013] [Accepted: 05/14/2013] [Indexed: 11/26/2022]
Abstract
Management of skin wound infections presents a serious problem in the clinic, in the community, and in both civilian and military clinical treatment centers. Staphylococcus aureus is one of the most common microbial pathogens in cutaneous wounds. Peptide-morpholino oligomer (PMO) conjugates targeted to S. aureus gyrase A mRNA have shown the ability to reduce bacterial viability by direct site-specific mRNA cleavage via RNase P. As a treatment, these conjugates have the added advantages of not being susceptible to resistance due to genetic mutations and are effective against drug resistant strains. While this strategy has proven effective in liquid culture, it has yet to be evaluated in an animal model of infected surface wounds. In the present study, we combined PMO conjugates with a thermoresponsive gel delivery system to treat full-thickness mouse cutaneous wounds infected with S. aureus. Wounds treated with a single dose of PMO conjugate displayed improved healing that was associated with increased epithelialization, reduced bacterial load, and increased matrix deposition. Taken together, our findings demonstrate the efficacy and flexibility of the PMO conjugate drug delivery system and make it an attractive and novel topical antimicrobial agent.
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Affiliation(s)
- Andrew J Sawyer
- Department of Pathology, Yale University, New Haven, CT 06520, USA
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Inhibition of cell division induced by external guide sequences (EGS Technology) targeting ftsZ. PLoS One 2012; 7:e47690. [PMID: 23110089 PMCID: PMC3479136 DOI: 10.1371/journal.pone.0047690] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2012] [Accepted: 09/18/2012] [Indexed: 01/20/2023] Open
Abstract
EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region “b” under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.
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12
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Cheng X, Ko JH, Altman S. Inactivation of expression of two genes in Saccharomyces cerevisiae with the external guide sequence methodology. RNA (NEW YORK, N.Y.) 2011; 17:544-9. [PMID: 21233222 PMCID: PMC3039153 DOI: 10.1261/rna.2538711] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/11/2010] [Accepted: 12/14/2010] [Indexed: 05/30/2023]
Abstract
The artificial inhibition of expression of genes in Saccharomyces cerevisiae is not a widespread, useful phenomenon. The external guide sequence (EGS) technology, which is well-proven in bacteria and mammalian cells in tissue culture and in mice, can also be utilized in yeast. The TOP2 and SRG1 genes can be inhibited by ∼30% with EGSs in vivo. Results in vitro also show convenient cleavage of the relevant transcripts by RNase P and appropriate EGSs. The feasible constructs shown to date have an EGS covalently linked to M1 RNA, the RNA subunit of RNase P from Escherichia coli. Greater efficiency in cleavage of transcripts can be fashioned using more than one EGS targeted to different sites in a transcript and stronger promoters controlling the EGS constructs.
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Affiliation(s)
- Xudong Cheng
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
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Abstract
The ability to interfere with gene expression is of crucial importance to unravel the function of genes and is also a promising therapeutic strategy. Here we discuss methodologies for inhibition of target RNAs based on the cleavage activity of the essential enzyme, Ribonuclease P (RNase P). RNase P-mediated cleavage of target RNAs can be directed by external guide sequences (EGSs) or by the use of the catalytic M1 RNA from E. coli linked to a guide sequence (M1GSs). These are not only basic tools for functional genetic studies in prokaryotic and eukaryotic cells but also promising antibacterial, anticancer and antiviral agents.
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Affiliation(s)
- Eirik Wasmuth Lundblad
- Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, 9038 Tromsø, Norway.
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Inhibition of aac(6')-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria. Proc Natl Acad Sci U S A 2009; 106:13230-5. [PMID: 19666539 DOI: 10.1073/pnas.0906529106] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.
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Generation of an external guide sequence library for a reverse genetic screen in Caenorhabditis elegans. BMC Biotechnol 2009; 9:47. [PMID: 19457250 PMCID: PMC2696436 DOI: 10.1186/1472-6750-9-47] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2008] [Accepted: 05/20/2009] [Indexed: 11/10/2022] Open
Abstract
Background A method for inhibiting the expression of particular genes using external guide sequences (EGSs) has been developed in bacteria, mammalian cells and maize cells. Results To examine whether EGS technology can be used to down-regulate gene expression in Caenorhabditis elegans (C. elegans), we generated EGS-Ngfp-lacZ and EGS-Mtgfp that are targeted against Ngfp-lacZ and Mtgfp mRNA, respectively. These EGSs were introduced, both separately and together, into the C. elegans strain PD4251, which contains Ngfp-lacZ and Mtgfp. Consequently, the expression levels of Ngfp-lacZ and Mtgfp were affected by EGS-Ngfp-lacZ and EGS-Mtgfp, respectively. We further generated an EGS library that contains a randomized antisense domain of tRNA-derived EGS ("3/4 EGS"). Examination of the composition of the EGS library showed that there was no obvious bias in the cloning of certain EGSs. A subset of EGSs was randomly chosen for screening in the C. elegans strain N2. About 6% of these EGSs induced abnormal phenotypes such as P0 slow postembryonic growth, P0 larval arrest, P0 larval lethality and P0 sterility. Of these, EGS-35 and EGS-83 caused the greatest phenotype changes, and their target mRNAs were identified as ZK858.7 mRNA and Lin-13 mRNA, respectively. Conclusion EGS technology can be used to down-regulate gene expression in C. elegans. The EGS library is a research tool for reverse genetic screening in C. elegans. These observations are potentially of great importance to further our understanding and use of C. elegans genomics.
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16
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Shan G. RNA interference as a gene knockdown technique. Int J Biochem Cell Biol 2009; 42:1243-51. [PMID: 19442757 DOI: 10.1016/j.biocel.2009.04.023] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2009] [Revised: 04/20/2009] [Accepted: 04/29/2009] [Indexed: 01/11/2023]
Abstract
Not many scientific breakthroughs bring significant advances simultaneously in both basic research and translational applications like the discovery of RNA interference. Along with the elucidation of the RNA interference pathway and the discovery of its participation in crucial biological events, a branch of science has grown to utilize the RNA interference pathway as a biotechnology for both basic and applied research. Small interference RNA, plasmid-, and virus-encoded short-hairpin RNA are now regular reagents in the tool box of biologists to knockdown the expression of specific genes posttranscriptionally. Efforts have also been made to develop RNA interference based therapeutics into reality. Many concerns about the RNA interference technique have now been answered through research and development, although hurdles are still present. In this review, the RNA interference/microRNA pathway is briefly introduced followed with a detailed summary about the design and application of the RNA interference experiments, along with examples of the utilization of the RNA interference technology in animal cells and model organisms. Recent progresses and current concerns are also highlighted. Two techniques, namely morpholino and external guide sequence, are discussed as complementary gene knockdown technology. RNA interference technology, along with several other alternative gene knockdown techniques, is now indispensable to modern biological and medical research.
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Affiliation(s)
- Ge Shan
- Department of Molecular, Cellular, and Developmental Biology, Yale University, P. O. Box 208103, New Haven, CT 06520, USA.
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Messenger RNA Turnover Processes in Escherichia coli, Bacillus subtilis, and Emerging Studies in Staphylococcus aureus. Int J Microbiol 2009; 2009:525491. [PMID: 19936110 PMCID: PMC2777011 DOI: 10.1155/2009/525491] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2008] [Accepted: 11/14/2008] [Indexed: 11/17/2022] Open
Abstract
The regulation of mRNA turnover is a recently appreciated phenomenon by which bacteria modulate gene expression. This review outlines the mechanisms by which three major classes of bacterial trans-acting factors, ribonucleases (RNases), RNA binding proteins, and small noncoding RNAs (sRNA), regulate the transcript stability and protein production of target genes. Because the mechanisms of RNA decay and maturation are best characterized in Escherichia coli, the majority of this review will focus on how these factors modulate mRNA stability in this organism. However, we also address the effects of RNases, RNA binding proteins, sRNAs on mRNA turnover, and gene expression in Bacillus subtilis, which has served as a model for studying RNA processing in gram-positive organisms. We conclude by discussing emerging studies on the role modulating mRNA stability has on gene expression in the important human pathogen Staphylococcus aureus.
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Rasmussen LCV, Sperling-Petersen HU, Mortensen KK. Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microb Cell Fact 2007; 6:24. [PMID: 17692125 PMCID: PMC1995221 DOI: 10.1186/1475-2859-6-24] [Citation(s) in RCA: 72] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2007] [Accepted: 08/10/2007] [Indexed: 12/16/2022] Open
Abstract
An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.
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Affiliation(s)
| | - Hans Uffe Sperling-Petersen
- Laboratory of BioDesign, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark
| | - Kim Kusk Mortensen
- Laboratory of BioDesign, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark
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19
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Ko JH, Altman S. OLE RNA, an RNA motif that is highly conserved in several extremophilic bacteria, is a substrate for and can be regulated by RNase P RNA. Proc Natl Acad Sci U S A 2007; 104:7815-20. [PMID: 17470803 PMCID: PMC1876530 DOI: 10.1073/pnas.0701715104] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
OLE (ornate, large, and extremophilic) RNA is a noncoding RNA that is found in several extremophilic bacteria, including Bacillus halodurans. The function of OLE RNA has not been clarified. In this study, we found that RNase P cleaves OLE RNA and that the cleavage leads to a small reduction of expression of a downstream gene determined by analyses in vitro and in vivo. Under RNase P-deficient conditions, the amount of OLE RNA increased. Our results imply that RNase P could play a role in the regulation of gene expression in relation to conserved RNA motifs like OLE RNA as well as in riboswitches and operons.
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Affiliation(s)
- Jae-hyeong Ko
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520
| | - Sidney Altman
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520
- *To whom correspondence may be addressed. E-mail:
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20
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Soler Bistué AJC, Ha H, Sarno R, Don M, Zorreguieta A, Tolmasky ME. External guide sequences targeting the aac(6')-Ib mRNA induce inhibition of amikacin resistance. Antimicrob Agents Chemother 2007; 51:1918-25. [PMID: 17387154 PMCID: PMC1891410 DOI: 10.1128/aac.01500-06] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The dissemination of AAC(6')-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6')-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6')-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6')-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 mug of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 mug of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin.
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Affiliation(s)
- Alfonso J C Soler Bistué
- Department of Biological Science, College of Natural Science and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA
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21
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Gao MY, Xu CR, Chen R, Liu SG, Feng JN. Chloromycetin resistance of clinically isolated E coli is conversed by using EGS technique to repress the chloromycetin acetyl transferase. World J Gastroenterol 2006; 11:7368-73. [PMID: 16437645 PMCID: PMC4725137 DOI: 10.3748/wjg.v11.i46.7368] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical E coli isolates from Cm- resistant to Cm- sensitive. METHODS EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene. The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl(2) transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E coli strains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones. The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A(600). Drug sensitivity was tested in solid culture containing Cm by using KB method. RESULTS Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 microg/mL of Cm) E coli strains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS. Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 microg/mL of Cm. In drug sensitivity test, these strains were sensitive to Cm on LB-agar plates containing 200 microg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones. CONCLUSION The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cm-resistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.
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Affiliation(s)
- Mei-Ying Gao
- Wuhan Bioproduct Institute of Ministry of Public Health, 9 Linjiang Dadao, Wuhan, 430030, Hubei Province, China
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22
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SU YZ, LI HJ, LI YQ, CHEN HJ, TANG DS, ZHANG X, JIANG H, ZHOU TH. In Vitro Construction of Effective M1GS Ribozymes Targeting HCMV UL54 RNA Segments. Acta Biochim Biophys Sin (Shanghai) 2005. [DOI: 10.1111/j.1745-7270.2005.00025.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
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23
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Abstract
RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.
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Affiliation(s)
- Phong Trang
- Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA
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24
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Abstract
Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy.
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Affiliation(s)
- Stephen M L Raj
- Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA 94720, USA
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25
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Abstract
Non-coding ribonucleic acids (RNAs) do not contain a peptide-encoding open reading frame and are therefore not translated into proteins. They are expressed in all phyla, and in eukaryotic cells they are found in the nucleus, cytoplasm, and mitochondria. Non-coding RNAs either can exert structural functions, as do transfer and ribosomal RNAs, or they can regulate gene expression. Non-coding RNAs with regulatory functions differ in size ranging from a few nucleotides to over 100 kb and have diverse cell- or development-specific functions. Some of the non-coding RNAs associate with human diseases. This chapter summarizes the current knowledge about regulatory non-coding RNAs.
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Affiliation(s)
- Uwe Michel
- Department of Neurology, Laboratory of Neurobiology, Göttingen, Germany
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26
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McKinney J, Guerrier-Takada C, Wesolowski D, Altman S. Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes. Proc Natl Acad Sci U S A 2001; 98:6605-10. [PMID: 11381134 PMCID: PMC34400 DOI: 10.1073/pnas.121180398] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.
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Affiliation(s)
- J McKinney
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA
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27
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Cole KB, Dorit RL. Protein cofactor-dependent acquisition of novel catalytic activity by the RNase P ribonucleoprotein of E. coli. J Mol Biol 2001; 307:1181-212. [PMID: 11292334 DOI: 10.1006/jmbi.2001.4519] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K(M). We trace the catalytic enhancement to two ubiquitous A-->U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency (k(cat)/K(M)). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.
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Affiliation(s)
- K B Cole
- Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, 06511, USA
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28
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In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment. Blood 2000. [DOI: 10.1182/blood.v95.3.731.003k28_731_737] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes againstBCR-ABLp190 andBCR-ABLp210, bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of theBCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.
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29
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Cole KB, Dorit RL. Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation. J Mol Biol 1999; 292:931-44. [PMID: 10525416 DOI: 10.1006/jmbi.1999.3098] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.
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Affiliation(s)
- K B Cole
- Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect St, New Haven, CT, 06511, USA
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30
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Kilani AF, Liu F. UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence. RNA (NEW YORK, N.Y.) 1999; 5:1235-1247. [PMID: 10496224 PMCID: PMC1369846 DOI: 10.1017/s1355838299990672] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.
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Affiliation(s)
- A F Kilani
- Program in Infectious Diseases and Immunity, University of California, Berkeley 94720, USA
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31
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Kawa D, Wang J, Yuan Y, Liu F. Inhibition of viral gene expression by human ribonuclease P. RNA (NEW YORK, N.Y.) 1998; 4:1397-406. [PMID: 9814760 PMCID: PMC1369712 DOI: 10.1017/s1355838298980918] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.
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MESH Headings
- Animals
- Base Sequence
- Blotting, Western
- Cells, Cultured
- Chlorocebus aethiops
- Endoribonucleases/genetics
- Endoribonucleases/metabolism
- Gene Expression Regulation, Viral
- Gene Targeting
- Herpesvirus 1, Human/drug effects
- Herpesvirus 1, Human/genetics
- Humans
- Molecular Sequence Data
- Mutation
- Oligonucleotides/genetics
- Oligonucleotides/pharmacology
- RNA, Catalytic/genetics
- RNA, Catalytic/metabolism
- RNA, Messenger/analysis
- RNA, Messenger/metabolism
- Ribonuclease P
- Ribonucleoproteins/genetics
- Ribonucleoproteins/metabolism
- Thymidine Kinase/genetics
- Transfection
- Vero Cells
- RNA, Small Untranslated
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Affiliation(s)
- D Kawa
- Program in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley 94720, USA
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32
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Hartmann RK, Krupp G, Hardt WD. Towards a new concept of gene inactivation: specific RNA cleavage by endogenous ribonuclease P. BIOTECHNOLOGY ANNUAL REVIEW 1998; 1:215-65. [PMID: 9704090 DOI: 10.1016/s1387-2656(08)70053-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
In the first part of this chapter, general concepts for gene inactivation, antisense techniques and catalytic RNAs (ribozymes) are presented. The requirements for modified oligonucleotides are discussed with their effects on the stability of base-paired hybrids and on resistance against nuclease attack. This also includes the problems in the choice of an optimal target sequence within the inactivated RNA and the options of cellular delivery systems. The second part describes the recently introduced antisense concept based on the ubiquitous cellular enzyme ribonuclease P. This system is unique, since the substrate recognition requires the proper tertiary structure of the cleaved RNA. General properties and possible advantages of this approach are discussed.
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Affiliation(s)
- R K Hartmann
- Institut für Biochemie, Freie Universität Berlin, Germany
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33
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Plehn-Dujowich D, Altman S. Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P. Proc Natl Acad Sci U S A 1998; 95:7327-32. [PMID: 9636148 PMCID: PMC22606 DOI: 10.1073/pnas.95.13.7327] [Citation(s) in RCA: 78] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/01/1998] [Indexed: 02/07/2023] Open
Abstract
The polymerase (PB2) and nucleocapsid (NP) genes encoded by the genome of influenza virus are essential for replication of the virus. When synthetic genes that express RNAs for external guide sequences targeted to the mRNAs of the PB2 and NP genes are stably incorporated into mouse cells in tissue culture, infection of these cells with influenza virus is nonproductive. Endogenous RNase P cleaves the targeted influenza virus mRNAs when they are in a complex with the external guide sequences. Targeting two different mRNAs simultaneously inhibits viral particle production more efficiently than does targeting only one mRNA.
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Affiliation(s)
- D Plehn-Dujowich
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
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34
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Guerrier-Takada C, Salavati R, Altman S. Phenotypic conversion of drug-resistant bacteria to drug sensitivity. Proc Natl Acad Sci U S A 1997; 94:8468-72. [PMID: 9238000 PMCID: PMC22959 DOI: 10.1073/pnas.94.16.8468] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Plasmids that contain synthetic genes coding for small oligoribonucleotides called external guide sequences (EGSs) have been introduced into strains of Escherichia coli harboring antibiotic resistance genes. The EGSs direct RNase P to cleave the mRNAs transcribed from these genes thereby converting the phenotype of drug-resistant cells to drug sensitivity. Increasing the EGS-to-target mRNA ratio by changing gene copy number or the number of EGSs complementary to different target sites enhances the efficiency of the conversion process. We demonstrate a general method for the efficient phenotypic conversion of drug-resistant bacterial cultures.
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35
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Liu F, Altman S. Requirements for cleavage by a modified RNase P of a small model substrate. Nucleic Acids Res 1996; 24:2690-6. [PMID: 8758997 PMCID: PMC145998 DOI: 10.1093/nar/24.14.2690] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, has been covalently linked at its 3' terminus to oligonucleotides (guide sequences) that guide the enzyme to target RNAs through hybridization with the target sequences. These constructs (M1GS RNAs) have been used to determine some minimal features of model substrates. As few as 3 bp on the 3' side of the site of cleavage in a substrate complex and 1 nt on the 5' side are required for cleavage to occur. The cytosines in the 3' terminal CCA sequence of the model substrates are important for cleavage efficiency but not cleavage site selection. A purine (base-paired or not) at the 3' side of the cleavage site is important both for cleavage site selection and efficiency. M1GS RNAs provide both a simple system for characterization of the reaction governed by M1 RNA and a tool for gene therapy.
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Affiliation(s)
- F Liu
- Department of Biology, Yale University, New Haven, CT 06520, USA
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36
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Li Y, Altman S. Cleavage by RNase P of gene N mRNA reduces bacteriophage lambda burst size. Nucleic Acids Res 1996; 24:835-42. [PMID: 8600449 PMCID: PMC145720 DOI: 10.1093/nar/24.5.835] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
RNase P, an enzyme essential for tRNA biosynthesis, can be directed to cleave any RNA when the target RNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS). RNase P from Escherichia coli can cleave phage lambda N mRNA in vitro or in vivo when the mRNA is in a complex with an EGS. The EGS can either be separate from or covalently linked to M1 RNA, the catalytic RNA subunit of RNase P. The requirement for Mg2+ in the reaction in vitro is lower when the EGS is covalently linked to M1 RNA. Substrates made of DNA can also be cleaved by RNase P in vitro in complexes with RNA EGSs. When either kind of EGS construct is used in vivo, burst size of phage lambda is reduced by > or = 40%. Reduction in burst size depends on efficient expression of the EGS constructs. The product of phage lambda gene N appears to function in a stoichiometric fashion.
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Affiliation(s)
- Y Li
- Department of Biology, Yale University, New Haven, CT 06520, USA
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37
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Kirsebom LA, Vioque A. RNase P from bacteria. Substrate recognition and function of the protein subunit. Mol Biol Rep 1996; 22:99-109. [PMID: 8901495 DOI: 10.1007/bf00988713] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5'-leader sequence as well as the 3'-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studied in vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.
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Affiliation(s)
- L A Kirsebom
- Department of Microbiology, Biomedical Center, Uppsala, Sweden
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38
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Guerrier-Takada C, Li Y, Altman S. Artificial regulation of gene expression in Escherichia coli by RNase P. Proc Natl Acad Sci U S A 1995; 92:11115-9. [PMID: 7479948 PMCID: PMC40582 DOI: 10.1073/pnas.92.24.11115] [Citation(s) in RCA: 84] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.
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39
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Altman S. RNase P in research and therapy. BIO/TECHNOLOGY (NATURE PUBLISHING COMPANY) 1995; 13:327-9. [PMID: 9634774 DOI: 10.1038/nbt0495-327] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Affiliation(s)
- S Altman
- Yale University, New Haven, CT 06511, USA
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40
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Liu F, Altman S. Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli. Genes Dev 1995; 9:471-80. [PMID: 7533740 DOI: 10.1101/gad.9.4.471] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by proteins extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by approximately 80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA.
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Affiliation(s)
- F Liu
- Department of Biology, Yale University, New Haven, Connecticut 06520
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41
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Sioud M, Opstad A, Zhao JQ, Levitz R, Benham C, Drlica K. In vivo decay kinetic parameters of hammerhead ribozymes. Nucleic Acids Res 1994; 22:5571-5. [PMID: 7838709 PMCID: PMC310118 DOI: 10.1093/nar/22.25.5571] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known. Recently, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s). To better understand the effect of different 3'-terminal hairpins on ribozyme stability as well as their effect on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed. One ribozyme contained a 3'-terminal hairpin derived from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay with a half life of approximately 250 h in all cases. The 3'-terminal hairpin has no significant effect on this component. This slow phase accounted for 60-80% of ribozyme decay. The rapid phase also exhibited exponential decay. For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4). The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme. Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).
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Affiliation(s)
- M Sioud
- Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway
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42
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Yuan Y, Altman S. Selection of guide sequences that direct efficient cleavage of mRNA by human ribonuclease P. Science 1994; 263:1269-73. [PMID: 8122108 DOI: 10.1126/science.8122108] [Citation(s) in RCA: 84] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Any RNA, when in a complex with another oligoribonucleotide known as an external guide sequence (EGS), can become a substrate for ribonuclease P. Simulation of evolution in vitro was used to select EGSs that bind tightly to a target substrate messenger RNA and that increase the efficiency of cleavage of the target by human ribonuclease P to a level equal to that achieved with natural substrates. The most efficient EGSs form transfer RNA precursor-like structures with the target RNA, in which the analog of the anticodon stem has been disrupted, an indication that selection for the optimal substrate for ribonuclease P yields an RNA structure different from that of present-day transfer RNA precursors.
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Affiliation(s)
- Y Yuan
- Department of Biology, Yale University, New Haven, CT 06520
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43
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Castanotto D, Rossi JJ, Sarver N. Antisense catalytic RNAs as therapeutic agents. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 1994; 25:289-317. [PMID: 8204504 DOI: 10.1016/s1054-3589(08)60435-4] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Affiliation(s)
- D Castanotto
- Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010
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Abstract
RNase P and RNase MRP are related ribonucleoproteins. RNase MRP processes mitochondrial precursor- (primer) RNAs, whereas RNase P cleaves precursor-tRNAs to produce their mature 5'-ends. Both RNase P and RNase MRP are associated with the Th/To ribonucleoprotein suggesting possible interrelated pathways and/or functions. All known RNase P and RNase MRP RNAs contain conserved structural elements possibly involved in catalysis/substrate binding, but these elements do not predict all cellular functions of the RNPs.
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Affiliation(s)
- R Karwan
- Institute of Tumor Biology, University of Vienna, Austria
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45
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Torrence PF, Maitra RK, Lesiak K, Khamnei S, Zhou A, Silverman RH. Targeting RNA for degradation with a (2'-5')oligoadenylate-antisense chimera. Proc Natl Acad Sci U S A 1993; 90:1300-4. [PMID: 7679499 PMCID: PMC45860 DOI: 10.1073/pnas.90.4.1300] [Citation(s) in RCA: 95] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases. However, targeted destruction of RNA has been difficult to achieve in a versatile, efficient, and reliable manner. We have developed an effective strategy for cleaving unique RNA sequences with 2-5A-dependent RNase, an endoribonuclease that mediates inhibitory effects of interferon on virus infection and is activated by 5'-phosphorylated 2'-5'-linked oligoadenylates known as 2-5A [pn5' A2'(p5' A2')mp5'A], resulting in the cleavage of single-stranded RNA predominantly after UpUp and UpAp sequences. To direct 2-5A-dependent RNase to cleave unique RNA sequences, p5' A2' p5' A2'p5'A was covalently linked to an antisense oligonucleotide to yield a chimeric molecule (2-5A:AS). The antisense oligonucleotide component of 2-5A:AS bound a specific RNA sequence while the accompanying 2-5A component activated 2-5A-dependent RNase, thereby causing the cleavage of the RNA in the targeted sequence. This strategy was demonstrated by inducing specific cleavage within a modified human immunodeficiency virus type 1 vif mRNA in a cell-free system from human lymphoblastoid cells. Because 2-5A-dependent RNase is present in most mammalian cells, the control of gene expression based on this technology--including therapies for cancer, viral infections, and certain genetic diseases--can be envisioned.
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Affiliation(s)
- P F Torrence
- Section on Biomedical Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
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46
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An indexed bibliography of antisense literature, 1992. ANTISENSE RESEARCH AND DEVELOPMENT 1993; 3:95-153. [PMID: 8495109 DOI: 10.1089/ard.1993.3.95] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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47
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Schlegl J, Fürste JP, Bald R, Erdmann VA, Hartmann RK. Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus. Nucleic Acids Res 1992; 20:5963-70. [PMID: 1281315 PMCID: PMC334461 DOI: 10.1093/nar/20.22.5963] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.
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Affiliation(s)
- J Schlegl
- Institut für Biochemie, Freie Universität Berlin, Germany
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48
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Abstract
Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.
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Affiliation(s)
- Y Yuan
- Department of Biology, Yale University, New Haven, CT 06520
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