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PubMed2004

Developing RNase P ribozymes for gene-targeting and antiviral therapy.

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.

Antiviral Agents↗

PubMed2004

General design and construction of RNase P ribozymes for gene-targeting applications.

RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, 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 M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence. Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications. Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions. Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included. These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.

Animals↗

PubMed1999

A computational screen for methylation guide snoRNAs in yeast.

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Many of the genes for this snoRNA family have remained unidentified in Saccharomyces cerevisiae, despite the availability of a complete genome sequence. Probabilistic modeling methods akin to those used in speech recognition and computational linguistics were used to computationally screen the yeast genome and identify 22 methylation guide snoRNAs, snR50 to snR71. Gene disruptions and other experimental characterization confirmed their methylation guide function. In total, 51 of the 55 ribose methylated sites in yeast ribosomal RNA were assigned to 41 different guide snoRNAs.

Algorithms↗

PubMed2006

Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

Base Sequence↗

PubMed2001

The cap-to-tail guide to mRNA turnover.

The levels of cellular messenger RNA transcripts can be regulated by controlling the rate at which the mRNA decays. Because decay rates affect the expression of specific genes, they provide a cell with flexibility in effecting rapid change. Moreover, many clinically relevant mRNAs--including several encoding cytokines, growth factors and proto-oncogenes--are regulated by differential RNA stability. But what are the sequence elements and factors that control the half-lives of mRNAs?

Animals↗

PubMed1987

Replicative events in hepatitis A virus-infected MRC-5 cells.

Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detectably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2-4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown.

Capsid↗

PubMed2026

RNA-repelling Anionic Clusters in Human Rhinovirus Cooperate with Cationic Residues to Promote Virion Assembly and Restrain RNA Release.

Research on virus nucleic acid-protein interactions is important to understand infection and guide antiviral drug design. In previous studies we showed that the human rhinovirus (RV) genomic RNA is organized as a dodecahedral cage formed by 30 RNA duplex elements anchored to capsid concavities. We showed also that capsid-RNA duplex interactions include conserved tryptophans, neutral polar residues, and many positively charged residues that promote virion assembly and restrain RNA release by stabilizing the negatively charged RNA duplex structure. The present study expands our understanding of the capsid-RNA duplex interface in RV by addressing the structural and functional roles of conserved patches of negatively charged capsid residues interposed between each RNA duplex and its binding site at the capsid inner surface. The initial hypothesis was that electrostatic repulsion between anionic residues and RNA phosphates would lead to functional effects opposite to those previously found for cationic residues that can electrostatically attract RNA phosphates. In fact, those anionic residues do not oppose, but act together with cationic residues at the RNA duplex binding sites to promote virion assembly and restrain RNA release. Cryogenic electron microscopy analysis showed that negatively charged residues at the capsid-RNA duplex interfaces have a different structural role than positively charged residues, even though they all play similar functional roles. A tentative model is discussed to explain the functional effects of the complex distribution of negative and positive electrostatic potential found at capsid-RNA duplex interfaces in RV.

capsid↗

PubMed2000

Diagnostic testing for hepatitis C.

Diagnostic tests for hepatitis C virus have improved dramatically over the past decade. Highly accurate tests are now available for screening patients for possible hepatitis C infections and confirming the presence of active viral infection. HCV RNA levels and genotype are very useful in assessing the likelihood of response to antiviral therapy and in guiding the optimum duration of treatment. Absence of detectable HCV RNA using PCR methodology has become the gold standard of successful treatment of patients with chronic hepatitis C. The various tests for hepatitis C are expensive and have their limitations. However, selective use of these assays has greatly improved the care of patients with chronic hepatitis C.

Antiviral Agents↗

PubMed2001

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Animals↗

PubMed2005

Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

Amino Acid Sequence↗

PubMed1998

Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents. Department of Health and Human Services and Henry J. Kaiser Family Foundation.

With the development and FDA approval of an increasing number of antiretroviral agents, decisions regarding the treatment of HIV-infected persons have become complex; and the field continues to evolve rapidly. In 1996, the Department of Health and Human Services and the Henry J. Kaiser Family Foundation convened the Panel on Clinical Practices for the Treatment of HIV to develop guidelines for the clinical management of HIV-infected persons. This report includes the guidelines developed by the Panel regarding the use of laboratory testing in initiating and managing antiretroviral therapy, considerations for initiating therapy, whom to treat, what regimen of antiretroviral agents to use, when to change the antiretroviral regimen, treatment of the acutely HIV-infected person, special considerations in adolescents, and special considerations in pregnant women. Viral load and CD4+ T cell testing should ideally be performed twice before initiating or changing an antiretroviral treatment regimen. All patients who have advanced or symptomatic HIV disease should receive aggressive antiretroviral therapy. Initiation of therapy in the asymptomatic person is more complex and involves consideration of multiple virologic, immunologic, and psychosocial factors. In general, persons who have <500 CD4+ T cells per mm3 should be offered therapy; however, the strength of the recommendation to treat should be based on the patient's willingness to accept therapy as well as the prognosis for AIDS-free survival as determined by the HIV RNA copy per mL of plasma and the CD4+ T cell count. Persons who have >500 CD4+ T cells per mm3 can be observed or can be offered therapy; again, risk of progression to AIDS, as determined by HIV RNA viremia and CD4+ T cell count, should guide the decision to treat. Once the decision to initiate antiretroviral therapy has been made, treatment should be aggressive with the goal of maximal viral suppression. In general, a protease inhibitor and two nucleoside [corrected] reverse transcriptase inhibitors should be used initially. Other regimens may be utilized but are considered less than optimal Many factors, including reappearance of previously undetectable HIV RNA, may indicate treatment failure. Decisions to change therapy and decisions regarding new regimens must be carefully considered; there are minimal clinical data to guide these decisions. Patients with acute HIV infection should probably be administered aggressive antiretroviral therapy; once initiated, duration of treatment is unknown and will likely need to continue for several years, if not for life. Special considerations apply to adolescents and pregnant women and are discussed in detail.

Adolescent↗

PubMed2001

Biogenesis of the Semliki Forest virus RNA replication complex.

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Animals↗

PubMed1990

The mechanism of group I self-splicing: an internal guide sequence can be provided in trans.

We have reconstituted a group I self-splicing reaction between two RNA molecules with different functional RNA parts: a substrate molecule containing the 5' splice site and a functional internal guide sequence (IGS), and a ribozyme molecule with core structure elements and splice sites but a mutated IGS. The 5' exon of the substrate molecule is ligated in trans to the 3' exon of the ribozyme molecule, suggesting that the deficient IGS in the ribozyme can be replaced by an externally added IGS present on the substrate molecule. This result is different from catalysis mediated by proteins where it is not possible to dissect the specificity of an enzyme from its catalytic activity.

Animals↗

PubMed2006

Coupling of transcription termination to RNAi.

In metazoans, the mechanisms of transcriptional termination by RNA polymerase II (Pol II) and accelerated decay of messenger RNA (mRNA) following transcription shutdown are linked by sharing the same sequence elements and mRNA elongation, processing and termination factors. This begs the question, how could one process have two opposite outcomes, making or degrading mRNA? An integrated "allosteric-GENEi-torpedo" model that could explain this paradox predicts participation of two novel factors: (1) An allosteric factor, regulated by a physiological repressor, binds to a unique sequence element of a gene near the site of cleavage and polyadenylation, poly(A) site, and acts on the homologous site on the nascent transcript to cause its cleavage. The conformational changes of this factor determine the fate of nascent RNA, either to get cleaved and processed to mature mRNA for directing protein synthesis, or not to get cleaved and become template for double-stranded (ds) RNA synthesis. (2) A general transcription termination factor, recruited by transcribing Pol II at the poly(A) site, allostrically alters and induces Pol II to switch template from DNA to nascent RNA several hundred nucleotides downstream of the poly(A) site. The template switch disengages Pol II from DNA and effectively terminates transcription. The Pol II with newly acquired RNA-dependent RNA polymerase activity retraces its path, back along the nascent RNA, so generating dsRNA. The extent to which it can retrace this path is determined by the factors influencing the cleavage of the pre-mRNA at the site of polyA addition. If cleavage and polyadenylation occur, the retracing is cut short, the 3' RNA is degraded by an exonuclease and the polymerase is liberated to reinitiate transcription. If the cleavage is inhibited, then a full-length dsRNA can be produced. This can then be subject to cleavage by "Dicer", which generates fragments of approximately 22bp that guide degradation of the cognate mRNA via the RNA interference (RNAi) pathway. This model complements the current "allosteric-torpedo" model of transcription termination, and could explain the apparent paradox of the divergent results of a common biological process.

DEAD-box RNA Helicases↗

PubMed1999

A Boltzmann filter improves the prediction of RNA folding pathways in a massively parallel genetic algorithm.

RNA folding using the massively parallel genetic algorithm (GA) has been enhanced by the addition of a Boltzmann filter. The filter uses the Boltzmann probability distribution in conjunction with Metropolis' relaxation algorithm. The combination of these two concepts within the GA's massively parallel computational environment helps guide the genetic algorithm to more accurately reflect RNA folding pathways and thus final solution structures. Helical regions (base-paired stems) now form in the structures based upon the stochastic properties of the thermodynamic parameters that have been determined from experiments. Thus, structural changes occur based upon the relative energetic impact that the change causes rather than just geometric conflicts alone. As a result, when comparing the predictions to phylogenetically determined structures, over multiple runs, fewer false-positive stems (predicted incorrectly) and more true-positive stems (predicted correctly) are generated, and the total number of predicted stems representing a solution is diminished. In addition, the significance (rate of occurrence) of the true-positive stems is increased. Thus, the predicted results more accurately reflect phylogenetically determined structures.

Algorithms↗

PubMed1999

UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

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.

Base Sequence↗

PubMed2006

Gene silencing with siRNA duplexes composed of target-mRNA-complementary and partially palindromic or partially complementary single-stranded siRNAs.

Synthetic small interfering RNA (siRNA) duplexes are widely used to transiently and sequence-specifically disrupt gene expression in mammalian cultured cells. The efficiency and specificity of mRNA cleavage is partly affected by the presence of the nontargeting "passenger" or "sense" siRNA strand, which is required for presentation of the target-complementary or guide siRNA strand to the double-strand-specific RNA silencing protein machinery. We show that siRNA duplexes can be designed that are solely composed of two fully target-complementary guide strands that are sufficiently complementary to each other to form stable duplexes with characteristic 3' overhanging ends. The general feasibility of this approach is documented by transient knockdown of lamin A/C and emerin in HeLa cells. The silencing efficiencies of guide-only siRNA duplexes are comparable to prototypical fully paired passenger/guide duplex siRNAs, even though guide-only siRNA duplexes may contain a significant number of nonWatson-Crick and G/U wobble base pairs. Such siRNA duplexes may offer advantages regarding production costs and specificity of gene silencing.

Base Sequence↗

PubMed2006

The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity.

The archaeal box C/D sRNP, the enzyme responsible for 2'-O-methylation of rRNA and tRNA, possesses a nearly perfect axis of symmetry and bipartite structure. This RNP contains two platforms for the assembly of protein factors, the C/D and C'/D' motifs, acting in conjunction with two guide sequences to direct methylation of a specific 2'-hydroxyl group in a target RNA. While this suggests that a functional asymmetric single-site complex complete with guide sequence and a single box C/D motif should be possible, previous work has demonstrated such constructs are not viable. To understand the basis for a bipartite RNP, we have designed and assayed the activity and specificity of a series of synthetic RNPs that represent a systematic reduction of the wild-type RNP to a fully single-site enzyme. This reduced RNP is active and exhibits all of the characteristics of wild-type box C/D RNPs except it is nonspecific with respect to the site of 2'-O-methylation. Our results demonstrate that protein-protein crosstalk through Nop5p dimerization is not required, but that architecture plays a crucial role in directing methylation activity with both C/D and C'/D' motifs being required for specificity.

Archaeal Proteins↗