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PubMed2006

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins.

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Amino Acid 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↗
PubMed2004

RNAi expression mediated inhibition of HCV replication.

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Cell Line, Tumor↗
PubMed2006

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

Binding Sites↗
PubMed1993

Triple aminoacylation specificity of a chimerized transfer RNA.

We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity. A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine. The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop. The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate. The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.

Acylation↗
PubMed1997

Control of gene expression in viruses and protozoan parasites by antisense oligonucleotides.

Chemically-modified oligonucleotides are now routinely used to prevent gene expression in cell-free media and in cultured cells. The binding of an antisense sequence to a complementary RNA target may lead to the selective inhibition of the encoded information. This may occur at different levels: splicing; transport of the mature RNA from the nucleus to the cytoplasm; translation. Antisense oligonucleotides constitute an interesting tool to shed some light on gene function. They are also potential new therapeutic agents against pathogenic organisms. This review discusses the rules that guide the design of an antisense oligomer and the choice of a target sequence. Examples of the potential use of antisense oligonucleotides in the fields of virology and parasitology, in particular in relation to trypanosomatids, are described.

Animals↗
PubMedDate not supplied

Genetic algorithm for the design of molecules with desired properties.

The design of molecules with desired properties is still a challenge because of the largely unpredictable end results. Computational methods can be used to assist and speed up this process. In particular, genetic algorithms have proved to be powerful tools with a wide range of applications, e.g. in the field of drug development. Here, we propose a new genetic algorithm that has been tailored to meet the demands of de novo drug design, i.e. efficient optimization based on small training sets that are analyzed in only a small number of design cycles. The efficiency of the design algorithm was demonstrated in the context of several different applications. First, RNA molecules were optimized with respect to folding energy. Second, a spinglass was optimized as a model system for the optimization of multiletter alphabet biopolymers such as peptides. Finally, the feasibility of the computer-assisted molecular design approach was demonstrated for the de novo construction of peptidic thrombin inhibitors using an iterative process of 4 design cycles of computer-guided optimization. Synthesis and experimental fitness determination of only 600 different compounds from a virtual library of more than 10(17) molecules was necessary to achieve this goal.

Algorithms↗
PubMed2004

Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

Antiviral Agents↗
PubMed2000

Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei. Sequence conservation and implications for 2'-O-ribose methylation of rRNA.

We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To systematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs. We have sequenced seventeen of them and designated them TBR for T. brucei RNA 1-17. All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes. Fourteen of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes.

Amino Acid Sequence↗
PubMed2007

Structure-guided peptidomimetic design leads to nanomolar beta-hairpin inhibitors of the Tat-TAR interaction of bovine immunodeficiency virus.

The Tat protein of immunodeficiency viruses is the main activator of viral gene expression. By binding specifically to its cognate site, the transactivator response element (TAR), Tat mediates a strong induction of the production of all viral transcripts. In seeking a new chemical solution to inhibiting viral protein-RNA interactions, we recently identified inhibitors of the viral Tat protein from the bovine immunodeficiency virus (BIV) using conformationally constrained beta-hairpin peptidomimetics. We identified a micromolar ligand, called BIV2, and the structure of its complex with BIV TAR was determined by NMR. In this work, we demonstrate that this chemistry can rapidly yield highly potent and selective ligands. On the basis of the structure, we synthesized and assayed libraries of mutant peptidomimetics. Remarkably, we were able in just a few rounds of design and synthesis to discover nanomolar inhibitors of the Tat-TAR interaction in BIV that selectively bind the BIV TAR RNA compared to RNA structures as closely related as the HIV-1 TAR or RRE elements. The molecular recognition principles developed in this study have been exploited in discovering related peptidomimetic inhibitors of the Tat-TAR interaction in HIV-1.

Amino Acid Sequence↗
PubMed1996

Structural features and stability of an RNA triple helix in solution.

A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.

Base Composition↗
PubMed1991

Site-directed mutagenesis of ricin A-chain and implications for the mechanism of action.

Ricin A-chain is an N-glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x-ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180-fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15- and 7-fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.

Base Sequence↗
PubMed2003

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila.

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.

Animals↗
PubMed2004

Biology of chronic myeloid leukemia and possible therapeutic approaches to imatinib-resistant disease.

Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the Bcr-Abl oncoprotein. An inhibitor of this tyrosine kinase, imatinib mesylate, is rapidly becoming the first-line therapy for CML. However, the development of resistance to this drug is a frequent setback, particularly in patients in advanced phases of the disease. Several mechanisms of resistance have been described, the most frequent of which are amplification and/or mutations of the BCR-ABL gene. To overcome resistance, several approaches have been studied in vitro and in vivo. They include dose escalation of imatinib, combination of imatinib with chemotherapeutic drugs, alternative Bcr-Abl inhibitors, inhibitors of kinases downstream of Bcr-Abl, farnesyl and geranylgeranyl transferase inhibitors, histone deacetylase, proteasome and cyclin-dependent kinase inhibitors, arsenic trioxide, hypomethylating agents, troxacitabine, targeting Bcr-Abl messenger RNA, and immunomodulatory strategies. It is important to understand that these approaches differ in efficiency, which is often dependent on the mechanisms of resistance. Further investigations into the molecular mechanisms of disease and how to specifically target the abnormal processes will guide the design of new treatment modalities in future clinical trials.

Antineoplastic Agents↗
PubMed2001

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

Binding, Competitive↗
PubMedDate not supplied

Antisense- and RNA interference-based therapeutic strategies in allergy.

Modern therapeutic methods for manipulation of gene expression in allergic diseases have been receiving increased attention in the emerging era of functional genomics. With the growing application of gene silencing technologies, pharmacological modulation of translation represents a great advance in molecular therapy for allergy. Several strategies for sequence-specific post-transcriptional inhibition of gene expression can be distinguished: antisense oligonucleotides (AS-ONs), ribozymes (RZs), DNA enzymes (DNAzymes), and RNA interference (RNAi) triggered by small interfering RNAs (siRNAs). Potential anti-mRNA drugs in asthma and other allergic disorders may be targeted to cell surface receptors (adenosine A1 receptor, high-affinity receptor Fc-epsilon RI-alpha, cytokine receptors), adhesion molecules and ligands (ICAM-1, VLA-4), ion channels (calcium-dependent chloride channel-1), cytokines and related factors (IL-4, IL-5, IL-13, SCF, TNF-alpha, TGF-beta1), intracellular signal transduction molecules, such as tyrosine-protein kinases (Syk, Lyn, Btk), serine/ threonine-protein kinases (p38 alpha MAPkinase, Raf-1), non-kinase signaling proteins (RasGRP4), and transcription factors involved in Th2 differentiation and allergic inflammation (STAT-6, GATA-3, NF-kappaB). The challenge to scientists is to determine which of the candidate targets warrants investment of time and resources. New-generation respirable AS-ONs, external guide sequence ribozymes, and RNA interference-based therapies have the potential to satisfy unmet needs in allergy treatment, acting at a more proximal level to a key etiopathogenetic molecular process, represented by abnormal expression of genes. Moreover, antisense and siRNA technologies imply a more rational design of new drugs for allergy.

Animals↗
PubMed2005

Uracil content of 16S rRNA of thermophilic and psychrophilic prokaryotes correlates inversely with their optimal growth temperatures.

We report here the finding of a highly significant inverse correlation of the uracil content of 16S rRNA and the optimum growth temperature (T(opt)) of cultured thermophilic and psychrophilic prokaryotes. This correlation was significantly different from the weaker correlations between the contents of other nucleotides and T(opt). Analysis of the 16S rRNA secondary structure regions revealed a fall in the A:U base-pair content in step with the increase in T(opt) that was much steeper than that of mismatched base-pairs, which are thermodynamically less stable. These findings indicate that the 16S rRNA sequences of thermophiles and psychrophiles are under a strong thermo-adaptive pressure, and that structure-function constraints play a crucial role in determining their 16S rRNA nucleotide composition. The derived relationship between uracil content and T(opt) was used to develop an algorithm to predict the T(opt) values of uncultured prokaryotes lacking cultured close relatives and belonging to the phyla predominantly containing thermophiles. This algorithm may be useful in guiding the design of cultivation conditions for hitherto uncultured microbes.

Adaptation, Physiological↗