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RNA Molecules (rna + molecule)
Selected AbstractsBiologically Important Reactions Catalyzed by RNA MoleculesTHE CHEMICAL RECORD, Issue 5 2002Yutaka Ikeda Abstract The last few years have seen a considerable increase in our understanding of catalysis by naturally occurring RNA molecules called ribozymes. The biological functions of RNA molecules depend upon their adoption of appropriate three-dimensional structures. The structure of RNA has a very important electrostatic component, which results from the presence of charged phosphodiester bonds. Metal ions are usually required to stabilize the folded structures and/or catalysis. Some ribozymes utilize metal ions as catalysts, whereas others use the ions to maintain appropriate three-dimensional structures. In the latter case, the correct folding of the RNA structures can perturb the pKa values of the nucleotide(s) within a catalytic pocket such that they act as general acid/bases catalysts. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 307,318, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10031 [source] Discovery of the hepatitis C virusLIVER INTERNATIONAL, Issue 2009Michael Houghton Abstract After nearly 6 years of intensive investigations between 1982 and 1988 in my laboratory at Chiron corporation, in which numerous molecular biological methods were used to investigate the viral aetiology of parenterally transmitted non-A, non-B viral hepatitis (NANBH), a single cDNA clone (5-1-1) was isolated that was shown to be derived from a new flavi-like virus, termed the hepatitis C virus (HCV). After screening hundreds of millions of bacterial cDNA clones derived from different liver and plasma samples obtained from experimentally infected chimpanzees, a single HCV clone was eventually isolated using a novel, blind immunoscreening method in which antibodies derived from a clinically diagnosed NANBH patient were used to identify a cDNA clone encoding an immunodominant epitope within HCV nonstructural protein 4. Its viral origin was demonstrated by its specific hybridization to a large single-stranded RNA molecule of ,10 000 nucleotides found only in NANBH-infected samples that shared distant sequence identity with flaviviruses. Further, HCV clone 5-1-1 was shown to be extrachromosomal and to encode an antigen eliciting antibody seroconversion only in NANBH-infected chimpanzees and humans. Subsequent work demonstrated that HCV was the principal cause of parenterally transmitted NANBH around the world, with an estimated 170 million global carriers and that blood screening tests detecting circulating HCV antibodies and viral RNA could effectively eradicate the transmission of transfusion-associated NANBH. Key viral-encoded enzymes essential to its life cycle are now the targets of vigorous, ongoing drug development activities, and the feasibility of successful vaccination strategies has been demonstrated using the valuable chimpanzee model, without which any progress on HCV would not have been possible. My colleagues and coworkers who made essential contributions to the discovery of HCV were George Kuo, who had his own laboratory at Chiron and who provided intellectual and practical input, Dan Bradley of the Centers for Disease Control and Prevention, who provided a large supply of well-characterized chimpanzee samples and knowledge of the NANBH field, and Qui-Lim Choo, in my own laboratory, who provided many years of outstandingly dedicated and precise molecular biology expertise. [source] DNA and RNA-Controlled Switching of Protein Kinase ActivityCHEMBIOCHEM, Issue 4 2009Lars Röglin Dr. Abstract Constrained: The readily programmable nucleic acid mediated recognition is used to constrain a phosphopeptide that was flanked by PNA segments. RNA-based switching allows control over the activity of target enzymes such as the protein kinase Src. It might thus be feasible to transduce changes of the concentration of selected RNA molecules to changes of the activity of signal transduction proteins. Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid,peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA,phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide,nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2,peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA,peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme. [source] Role and therapeutic potential of microRNAs in diabetesDIABETES OBESITY & METABOLISM, Issue 2009I. G. M. Kolfschoten The discovery in mammalian cells of hundreds of small RNA molecules, called microRNAs, with the potential to modulate the expression of the majority of the protein-coding genes has revolutionized many areas of biomedical research, including the diabetes field. MicroRNAs function as translational repressors and are emerging as key regulators of most, if not all, physiological processes. Moreover, alterations in the level or function of microRNAs are associated with an increasing number of diseases. Here, we describe the mechanisms governing the biogenesis and activities of microRNAs. We present evidence for the involvement of microRNAs in diabetes mellitus, by outlining the contribution of these small RNA molecules in the control of pancreatic ,-cell functions and by reviewing recent studies reporting changes in microRNA expression in tissues isolated from diabetes animal models. MicroRNAs hold great potential as therapeutic targets. We describe the strategies developed for the delivery of molecules mimicking or blocking the function of these tiny regulators of gene expression in living animals. In addition, because changes in serum microRNA profiles have been shown to occur in association with different human diseases, we also discuss the potential use of microRNAs as blood biomarkers for prevention and management of diabetes. [source] Regulation of miRNA expression during neural cell specificationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Lena Smirnova Abstract MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. We have studied the regulation of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. We observed significant differences in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes we found marked lineage specificity for each of the miRNA in our study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir-124, mir-128). In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. mir-26 and mir-29 were more strongly expressed in astrocytes than neurons, others were more evenly distributed (mir-9, mir-125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir-125 and mir-128). miRNA-mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA were strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development. [source] Phylogenetic distribution of microRNAs supports the basal position of acoel flatworms and the polyphyly of PlatyhelminthesEVOLUTION AND DEVELOPMENT, Issue 5 2007Lorenzo F. Sempere SUMMARY Phylogenetic analyses based on gene sequences suggest that acoel flatworms are not members of the phylum Platyhelminthes, but instead are the most basal branch of triploblastic bilaterians. Nonetheless, this result has been called into question. An alternative test is to use qualitative molecular markers that should, in principle, exclude the possibility of convergent (homoplastic) evolution in unrelated groups. microRNAs (miRNAs), noncoding regulatory RNA molecules that are under intense stabilizing selection, are a newly discovered set of phylogenetic markers that can resolve such taxonomic disputes. The acoel Childia sp. has recently been shown to possess a subset of the conserved core of miRNAs found across deuterostomes and protostomes, whereas a polyclad flatworm,in addition to this core subset,possesses miRNAs restricted to just protostomes. Here, we examine another acoel, Symsagittifera roscoffensis, and three other platyhelminths. Our results show that the distribution of miRNAs in S. roscoffensis parallels that of Childia. In addition, two of 13 new miRNAs cloned from a triclad flatworm are also found in other lophotrochozoan protostomes, but not in ecdysozoans, deuterostomes, or in basal metazoans including acoels. The limited set of miRNAs found in acoels, intermediate between the even more reduced set in cnidarians and the larger and expanding set in the rest of bilaterians, is compelling evidence for the basal position of acoel flatworms and the polyphyly of Platyhelminthes. [source] Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activityFEBS JOURNAL, Issue 7 2001Constantinos Stathopoulos Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5,-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment. [source] The basidiomycete genus Polyporus , an emendation based on phylogeny and putative secondary structure of ribosomal RNA moleculesFEDDES REPERTORIUM, Issue 7-8 2004D. Krüger The fungal genus Polyporus is an assemblage of white-rotting lignicolous basidiomycetes. It has undergone considerable expansion and contraction over a period of two and three quarter centuries. Current generic circumscription of Polyporus has kept the genus non-monophyletic. Species of Polyporus infrageneric group Polyporellus are closely related to some species of Lentinus. We introduce data for ITS2 spacer rRNA secondary structure evolution by quasi-independent comparison with large subunit rRNA phylogeny, and suggest a fraction of primary nuclear rDNA ITS sequence data as novel taxonomic character. A major taxonomic shift is suggested, supported by molecular and morphological characters, and allowing inclusion of species with gilled hymenophores in Polyporus. Two new names are proposed: Polyporus phyllostipes D.Krüger, nom. nov. and Polyporus gerdai D.Krüger, nom. nov. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Die Gattung Polyporus (Basidiomycetes) , eine Emendation auf der Basis von Phylogenie und mutmaßlicher sekundärer Struktur der ribosomalen RNA-Moleküle Die Pilzgattung Polyporus, eine Gruppe Weißfäule erregender holzbewohnender Basidiomyceten, wurde über nahezu drei Jahrhunderte vielfach expandiert und verkleinert. Bei der derzeitigen Gattungsumschreibung von Polyporus gilt die Gattung als nicht-monophyletisch. Arten der Polyporus -Gruppe Polyporellus sind eng verwandt zu einigen Lentinus- Arten. Anhand quasi-unabhängigem Vergleich mit der Phylogenie der rRNA der großen Untereinheit (LSU) stellen wir Daten zur Evolution der ITS2 Spacer rRNA vor, und schlagen ein ITS Kern-rDNA-Fragment als taxonomisches Merkmal vor. Unterstützt mit molekularen und morphologischen Daten wird eine grundlegende taxonomische Verschiebung vorgeschlagen, welche Arten mit Lamellen-Hymenophoren in Polyporus erlaubt. Zwei neue Namen werden vorgeschlagen: Polyporus phyllostipes D.Krüger, nom. nov. und Polyporus gerdai D.Krüger, nom. nov. [source] Rubber tree (Hevea brasiliensis) trunk phloem necrosis: aetiological investigations failed to confirm any biotic causal agentFOREST PATHOLOGY, Issue 1 2007F. Pellegrin Summary Trunk phloem necrosis (TPN) is currently a main constraint in rubber (Hevea brasiliensis) plantations. The apparent spread of the disease, from tree to tree along the planting line, strongly supported the implication of a pathogen that could be transmitted mechanically via the tapping knife. In order to detect a causal agent of the disease, studies focusing on characterization of the known mechanically transmitted pathogens (e.g. viroids, cryptic viruses or phytoplasma) were initiated. RNA strands of low molecular weight (200,400 and >500 bp) displaying structural similarities with viroids and viral dsRNAs were observed in various tested samples. However, attempts to show the potential role of these RNA molecules in the spread of the disease failed. First of all, there was no significant or reproducible correlation between the health status of the rubber trees sampled and these RNA molecules. Moreover, no sequence homology with known pathogens could be found when randomly amplified cDNA fragments isolated from trees presenting the disease symptoms were sequenced. In conclusion, the aetiological investigations, in order to show the presence of a pathogen responsible of the TPN disease, were non-conclusive, which tends to disprove the hypothesis of a biotic causal agent. [source] microRNAs in acute myeloid leukemia: Expression patterns, correlations with genetic and clinical parameters, and prognostic significanceGENES, CHROMOSOMES AND CANCER, Issue 3 2010Rotraud Wieser Acute myeloid leukemia (AML) is a malignant disease of hematopoietic cells whose emergence, course, and prognosis is affected by specific recurrent genetic alterations like chromosome aberrations and point mutations, as well as by changes in the expression of certain genes. In the past 2 years, microRNAs (miRNAs),a novel class of small RNA molecules involved in posttranscriptional gene regulation,have also been shown to be aberrantly expressed in AML. Furthermore, specific miRNA expression patterns were found to be associated with certain genetic and cytogenetic alterations in this disease, and two studies identified miRNAs whose expression levels were predictive of survival. Interestingly, the results of these analyses showed only very limited congruence. This review summarizes published reports on the expression patterns of miRNAs in AML, and discusses possible reasons for the differences in their results. © 2009 Wiley-Liss, Inc. [source] RNase P RNA-mediated cleavageIUBMB LIFE, Issue 3 2009Leif A. Kirsebom Abstract Metal(II)-induced hydrolysis of RNA produce products with 5,-hydroxyls and 2,;3,-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5,-hydroxyls and 2,;3,-cyclic phosphates whereas others produces 5,-phosphates and 3,-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5,-phosphates and 3,-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg2+. RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg2+ at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective. © 2009 IUBMB IUBMB Life, 61(3):189,200, 2009 [source] Inhibition of Lamin A/C Attenuates Osteoblast Differentiation and Enhances RANKL-Dependent Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2009Martina Rauner Abstract Age-related osteoporosis is characterized by low bone mass, poor bone quality, and impaired osteoblastogenesis. Recently, the Hutchinson-Gilford progeria syndrome (HGPS), a disease of accelerated aging and premature osteoporosis, has been linked to mutations in the gene encoding for the nuclear lamina protein lamin A/C. Here, we tested the hypothesis that inhibition of lamin A/C in osteoblastic lineage cells impairs osteoblastogenesis and accelerates osteoclastogenesis. Lamin A/C was knocked-down with small interfering (si)RNA molecules in human bone marrow stromal cells (BMSCs) differentiating toward osteoblasts. Lamin A/C knockdown led to an inhibition of osteoblast proliferation by 26% and impaired osteoblast differentiation by 48% based on the formation of mineralized matrix. In mature osteoblasts, expression levels of runx2 and osteocalcin mRNA were decreased by lamin A/C knockdown by 44% and 78%, respectively. Furthermore, protein analysis showed that osteoblasts with diminished levels of lamin A/C also secreted less osteocalcin and expressed a lower alkaline phosphatase activity (,50%). Lamin A/C inhibition increased RANKL mRNA and protein levels, whereas osteoprotegerin (OPG) expression was decreased, resulting in an increased RANKL/OPG ratio and an enhanced ability to support osteoclastogenesis, as reflected by a 34% increase of TRACP+ multinucleated cells. Our data indicate that lamin A/C is essential for proper osteoblastogenesis. Moreover, lack of lamin A/C favors an osteoclastogenic milieu and contributes to enhanced osteoclastogenesis. [source] Antisense applications for biological controlJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Wei-Hua Pan Abstract Although Nature's antisense approaches are clearly impressive, this Perspectives article focuses on the experimental uses of antisense reagents (ASRs) for control of biological processes. ASRs comprise antisense oligonucleotides (ASOs), and their catalytically active counterparts ribozymes and DNAzymes, as well as small interfering RNAs (siRNAs). ASOs and ribozymes/DNAzymes target RNA molecules on the basis of Watson-Crick base pairing in sequence-specific manner. ASOs generally result in destruction of the target RNA by RNase-H mediated mechanisms, although they may also sterically block translation, also resulting in loss of protein production. Ribozymes and DNAzymes cleave target RNAs after base pairing via their antisense flanking arms. siRNAs, which contain both sense and antisense regions from a target RNA, can mediate target RNA destruction via RNAi and the RISC, although they can also function at the transcriptional level. A considerable number of ASRs (mostly ASOs) have progressed into clinical trials, although most have relatively long histories in Phase I/II settings. Clinical trial results are surprisingly difficult to find, although few ASRs appear to have yet established efficacy in Phase III levels. Evolution of ASRs has included: (a) Modifications to ASOs to render them nuclease resistant, with analogous modifications to siRNAs being developed; and (b) Development of strategies to select optimal sites for targeting. Perhaps the biggest barrier to effective therapies with ASRs is the "Delivery Problem." Various liposomal vehicles have been used for systemic delivery with some success, and recent modifications appear to enhance systemic delivery, at least to liver. Various nanoparticle formulations are now being developed which may also enhance delivery. Going forward, topical applications of ASRs would seem to have the best chances for success. In summary, modifications to ASRs to enhance stability, improve targeting, and incremental improvements in delivery vehicles continue to make ASRs attractive as molecular therapeutics, but their advance toward the bedside has been agonizingly slow. J. Cell. Biochem. 98: 14,35, 2006. © 2006 Wiley-Liss, Inc. [source] Optimized transfection of diced siRNA into mature primary human osteoclasts: Inhibition of cathepsin K mediated bone resorption by siRNAJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005Christina I. Selinger Abstract Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by ,60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P,=,0.018) and resorbed area (P,=,0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects. © 2005 Wiley-Liss, Inc. [source] Minimum sequence requirements for selective RNA-ligand binding: A molecular mechanics algorithm using molecular dynamics and free-energy techniquesJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 14 2006Peter C. Anderson Abstract In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology. © 2006 Wiley Periodicals, Inc. J Comput Chem, 2006 [source] Reduction of Dicer impairs Schwann cell differentiation and myelinationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2010Jonathan D. Verrier Abstract The process of Schwann cell myelination requires precisely coordinated gene expression. At the onset of myelination, there is an increase in the expression of differentiation-promoting transcription factors that regulate key Schwann cell genes. Further control of myelin gene expression occurs at the posttranscriptional level and, in part, is mediated by RNA binding proteins and micro-RNAs (miRNAs). miRNAs are small, endogenously derived RNA molecules that repress gene expression by specifically binding to their mRNA targets. In the experiments described here, we tested whether miRNAs were essential in controlling myelination by reducing the levels of Dicer, an essential endoribonuclease in miRNA biogenesis. We decreased the expression of Dicer by about 60% within Schwann cells using a lentiviral vector expressing an shRNA against Dicer. The reduced levels of Dicer led to a decrease in the steady-state expression of selected miRNAs and of the transcription factors Oct6 and Egr2/Krox20, both of which are critical for Schwann cells differentiation and myelination. In contrast, the levels of c-jun and Sox2 were up-regulated by the reduction in Dicer and were associated with an increase in Schwann cell proliferation. In dorsal root ganglion cocultures, Schwann cells transduced with Dicer shRNA synthesized less myelin, which was accompanied by significant reductions in the levels of myelin basic protein and protein zero. These findings support a critical role for Dicer and miRNAs in Schwann cell differentiation and myelination. © 2010 Wiley-Liss, Inc. [source] Microarray profile of micro-ribonucleic acid in tumor tissue from cervical squamous cell carcinoma without human papillomavirusJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 5 2009YanLiang Zhang Abstract Aims:, Micro-ribonucleic acid (miRNA) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNA play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases. This study describes a comparison between the microRNA profile of human-papillomavirus-negative cervical squamous cell carcinoma patients and controls, in order to develop further understanding of the pathogenesis of cervical squamous cell carcinomas. Methods:, MiRNA were isolated from tumor tissues of five human-papillomavirus-negative cervical squamous cell carcinoma patients and five healthy controls in order to perform miRNA microarray chip analysis. The chip results were then confirmed by northern blot analysis. Results:, A total of 27 miRNA differentially expressed between the squamous cell carcinoma patients and the healthy controls were identified. Conclusion:, This work indicates that these miRNA may be potential diagnosis biomarkers and probable factors involved in the pathogenesis of cervical squamous cell carcinomas. [source] Full genomic amplification and subtyping of influenza A virus using a single set of universal primersMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2010Emi Inoue ABSTRACT Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes. [source] RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA constructMOLECULAR PLANT PATHOLOGY, Issue 4 2009NORA SCHWIND SUMMARY Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (,)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses. [source] Potato spindle tuber viroid: the simplicity paradox resolved?MOLECULAR PLANT PATHOLOGY, Issue 5 2007ROBERT A. OWENS SUMMARY Taxonomy: , Potato spindle tuber viroid (PSTVd) is the type species of the genus Posipiviroid, family Pospiviroidae. An absence of hammerhead ribozymes and the presence of a ,central conserved region' distinguish PSTVd and related viroids from members of a second viroid family, the Avsunviroidae. Physical properties: , Viroids are small, unencapsidated, circular, single-stranded RNA molecules which replicate autonomously when inoculated into host plants. Because viroids are non-protein-coding RNAs, designation of the more abundant, highly infectious polarity strand as the positive strand is arbitrary. PSTVd assumes a rod-like, highly structured conformation that is resistant to nuclease degradation in vitro. Naturally occurring sequence variants of PSTVd range in size from 356 to 361 nt. Hosts and symptoms: , The natural host range of PSTVd,cultivated potato, certain other Solanum spp., and avocado,appears to be quite limited. Foliar symptoms in potato are often obscure, and the severity of tuber symptoms (elongation with the appearance of prominent bud scales/eyebrows and growth cracks) depends on both temperature and length of infection. PSTVd has a broad experimental host range, especially among solanaceous species, and strains are classified as mild, intermediate or severe based upon the symptoms observed in sensitive tomato cultivars. These symptoms include shortening of internodes, petioles and mid-ribs, severe epinasty and wrinkling of the leaves, and necrosis of mid-ribs, petioles and stems. [source] Rust of flax and linseed caused by Melampsora liniMOLECULAR PLANT PATHOLOGY, Issue 4 2007GREGORY J. LAWRENCE SUMMARY Melampsora lini, while of economic importance as the causal agent of rust disease of flax and linseed, has for several decades been the ,model' rust species with respect to genetic studies of avirulence/virulence. Studies by Harold Flor demonstrated that single pairs of allelic genes determine the avirulence/virulence phenotype on host lines with particular resistance genes and led him to propose his famous ,gene-for-gene' hypothesis. Flor's inheritance studies, together with those subsequently carried out by others, also revealed that, in some cases, an inhibitor gene pair and an avirulence/virulence gene pair interact to determine the infection outcome on host lines with particular resistance genes. Recently, avirulence/virulence genes at four loci, AvrL567, AvrM, AvrP4 and AvrP/AvrP123, have been cloned. All encode novel, small, secreted proteins that are recognized inside plant cells. Yeast two-hybrid studies have shown that the AvrL567 proteins interact directly with the resistance gene protein. The molecular basis of Flor's gene-for-gene relationship has now been elucidated for six interacting gene pairs: those involving resistance genes L5, L6, L7, M, P and P2, where both the resistance gene and the corresponding avirulence gene have been cloned. In other inheritance studies it has been shown that M. lini does not possess a (+) and (,) mating system, but may possess a two factor system. Double-stranded (ds) RNA molecules occur in many strains of M. lini: examination of the progeny of one strain that possesses 11 dsRNA molecules revealed that they fall into three transmission units, designated L, A and B. The L unit consists of a single large dsRNA of 5.2 kbp while the A and B units each consist of five dsRNAs in the size range 1.1,2.8 kbp. The three units have different sexual and asexual transmission characteristics. The L unit is encapsidated in a virus-like particle, whereas the other units are not encapsidated. The population and coevolutionary aspects of M. lini on a wild, native Australian host species, Linum marginale, have been extensively investigated. A recent molecular analysis revealed that the M. lini isolates from L. marginale fall into two distinct lineages, one of which is apparently hybrid between two diverse genomes. Isolates in this lineage are largely fixed for heterozygosity, which suggests that sexual recombination does not occur in this lineage. [source] The impact of miR-34a on protein output in hepatocellular carcinoma HepG2 cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2010Jun Cheng Abstract MicroRNAs are small non-coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR-34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR-34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR-34a. Transfection of miR-34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2-DE method, 34 proteins were successfully identified by MALDI-TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR-34a. Bioinformatics analysis produced a protein,protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR-34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR-34a. In conclusion, abrogation of miR-34a function could cause downstream molecules to switch on or off, leading to HCC development. [source] Biologically Important Reactions Catalyzed by RNA MoleculesTHE CHEMICAL RECORD, Issue 5 2002Yutaka Ikeda Abstract The last few years have seen a considerable increase in our understanding of catalysis by naturally occurring RNA molecules called ribozymes. The biological functions of RNA molecules depend upon their adoption of appropriate three-dimensional structures. The structure of RNA has a very important electrostatic component, which results from the presence of charged phosphodiester bonds. Metal ions are usually required to stabilize the folded structures and/or catalysis. Some ribozymes utilize metal ions as catalysts, whereas others use the ions to maintain appropriate three-dimensional structures. In the latter case, the correct folding of the RNA structures can perturb the pKa values of the nucleotide(s) within a catalytic pocket such that they act as general acid/bases catalysts. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 307,318, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10031 [source] REVIEW ARTICLE: The Role of Placental Exosomes in ReproductionAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2010Lucia Mincheva-Nilsson Citation Mincheva-Nilsson L, Baranov V. The Role of Placental Exosomes in Reproduction. Am J Reprod Immunol 2010 Cell communication comprises cell,cell contact, soluble mediators and intercellular nanotubes. There is, however, another cell,cell communication by released membrane-bound microvesicles that convey cell,cell contact ,by proxy' transporting signals/packages of information from donor to recipient cells locally and/or at a distance. The nanosized exosomes comprise a specialized type of microvesicles generated within multivesicular bodies (MVB) and released upon MVB fusion with the plasma membrane. Exosomes are produced by a variety of immune, epithelial and tumor cells. Upon contact, exosomes transfer molecules that can render new properties and/or reprogram their recipient cells. Recently, it was discovered that the syncytiotrophoblast constitutively and throughout the pregnancy secretes exosomes. The placenta-derived exosomes are immunosuppressive and carry proteins and RNA molecules that in a redundant way influence a number of mechanisms and promote the fetal allograft survival. In this review, we summarize the current knowledge on the nature of placenta-derived exosomes and discuss their role in pregnancy. [source] Expression patterns and association analysis of the porcine DHX58 geneANIMAL GENETICS, Issue 5 2010X. Y. Li Summary RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH (Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of 1ipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron, was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine. [source] MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencingANIMAL GENETICS, Issue 2 2010M. Nielsen Summary MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3, untranslated region (3, UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5, and 3, ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function. [source] Requirement of toll-like receptor 7 for pristane-induced production of autoantibodies and development of murine lupus nephritis,ARTHRITIS & RHEUMATISM, Issue 4 2008Emina Savarese Objective The detection of high titers of antibodies against small nuclear ribonucleoproteins (snRNP) is a diagnostic finding in patients in whom systemic lupus erythematosus (SLE) is suspected. Endogenous RNA molecules within snRNP trigger Toll-like receptor 7 (TLR-7) activation in B cells and dendritic cells, leading to anti-snRNP antibody production, which is associated with the development of immune complex nephritis in SLE. The purpose of this study was to investigate the role of TLR-7 in anti-snRNP antibody production and renal disease in SLE induced by an exogenous factor in the absence of genetic predisposition, using the pristane-induced murine lupus model. Methods Serum autoantibodies, IgG isotypes, and cytokine levels in pristane-treated wild-type and TLR-7,deficient mice were analyzed by enzyme-linked immunosorbent assay. Histopathologic changes in mouse kidneys were determined by light immunofluorescence microscopy. Cell subsets in splenocytes and peritoneal lavage cells from the mice were examined by flow cytometry. Results We found that anti-snRNP antibody production induced by pristane treatment was entirely dependent on the expression of TLR-7, whereas anti,double-stranded DNA antibody production was not affected by a lack of TLR-7. Impaired anti-snRNP antibody production in TLR-7,deficient mice was paralleled by lower levels of glomerular IgG and complement deposits, as well as less severe glomerulonephritis. Conclusion TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis in pristane-induced murine lupus, a model of environmentally triggered SLE in the absence of genetic susceptibility to autoimmunity. Specific interference with TLR-7 activation by endogenous TLR-7 ligands may therefore be a promising novel strategy for the treatment of SLE. [source] Structure of the ribosomal protein L1,mRNA complex at 2.1,Å resolution: common features of crystal packing of L1,RNA complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2006S. Tishchenko The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1,Å resolution. It was found that all polar atoms involved in conserved protein,RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K+ ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1,RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open,open or open,closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation. [source] Prions at the crossroads: the need to identify the active TSE agentBIOESSAYS, Issue 5 2004P.K. Nandi Structural change in the cellular prion protein, PrPC to a ProteinaseK-resistant ,-sheet-rich insoluble form PrPSC and its accumulation have been considered to be central to the pathogenesis of the prion diseases (TSE). In a recent paper, Deleault et al have shown that specific endogenous RNA molecules can induce in vitro structural conversion of endogenous PrPC to PrPSC.1 Small highly structured synthetic RNAs can also induce this conversion process.2 However, recent in vivo results show that PrPSC is not directly involved in the prion pathogenesis.3 It is possible, however, that nucleic-acid-induced PrPSC associated with the inducer nucleic acid could be the components of the infectious agent. BioEssays 26:469,473, 2004. © 2004 Wiley Periodicals, Inc. [source] Trans -splicing in DrosophilaBIOESSAYS, Issue 11 2002Vincenzo Pirrotta Splicing is an efficient and precise mechanism that removes noncoding regions from a single primary RNA transcript. Cutting and rejoining of the segments occurs on nascent RNA. Trans -splicing between small specialized RNAs and a primary transcript has been known in some organisms but recent papers show that trans -splicing between two RNA molecules containing different coding regions is the normal mode in a Drosophila gene.1,3 The mod(mdg4) gene produces 26 different mRNAs encoding as many protein isoforms. The differences lie in alternative 3, exons encoded by different transcriptional units and spliced to the 5, common region by a surprising trans -splicing mechanism. BioEssays 24:988,991, 2002. © 2002 Wiley-Periodicals, Inc. [source] | |||||||||||||||||||||||||||||||