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At least 163 records · Page 9Linked to original sources
PubMed2002

RNA interference in the pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. This organism has recently been developed as an experimental system, with initiation of a genome project among other molecular advances. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. We have shown that expression of double-stranded RNA corresponding to portions of the cryptococcal CAP59 and ADE2 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Specific modulation of protein expression through introduction of double-stranded RNA thus operates in C. neoformans, which is the first demonstration of this technique in a fungal organism. Use of RNA interference in Cryptococcus should allow manipulation of mRNA levels for functional analysis of genes of interest and enable efficient exploration of genes discovered by genome sequencing.

Carboxy-Lyases↗
PubMedDate not supplied

Cytotoxic ribonucleases and RNA interference (RNAi).

Several cytotoxic ribonucleases (CRs), homologs of the pancreatic RNase A, have been isolated from amphibian oocytes or embryos. Of them, onconase (Onc), the CR that shows antitumor properties and is in phase III clinical trials, was the most extensively researched. Degradation of tRNA by Onc internalized into cells that leads to inhibition of protein synthesis is considered the mechanism of its cytotoxicity. Several findings, however, cannot be explained by nonspecific decline in protein synthesis alone and suggest additional or alternative mechanism(s). We postulate therefore that miRNAs and/or RNA interference (RNAi) may also be targets of CRs. The following arguments support this postulate: (A) miRNAs and siRNAs appear to be unprotected by proteins and therefore, as tRNA, accessible and degradable by CRs; (B) Onc has preferred cleavage sites on tRNAs: their cleavage may generate segments of dsRNA that interfere with translation. Analogous to Dicer, thus, small RNAs with interfering properties may be generated by CRs within the cell; (C) CRs are abundant in oocytes and during embryonic development; their role there is unknown. Since cells undergo perpetual differentiation during embryogenesis it is likely that the function of CRs is to provide additional level of regulation of gene expression via the mechanisms listed in (A) and/or (B).

Animals↗
PubMed2003

RNA interference as a metabolic engineering tool: potential for in vivo control of protein expression in an insect larval model.

Many ex vivo factors influence the yield of recombinant protein produced via AcMNPV (Autographa californica multiple nucleocapsid nuclear polyhedrosis virus) in Trichoplusia ni (T. ni) larvae. Among these are: the method of infection, the time of infection, the virus load, and the time of harvest. In vivo strategies, however, that attempt to manipulate host function in this and other expression systems have largely been ignored. In this work, RNA interference (RNAi) is shown as an effective metabolic engineering controller to downregulate targeted gene expression. Specifically, RNAi was made to virus-encoded gfp(uv) and was found to inhibit the production of GFPuv in larvae when injected within an 18-h window (before and after) of baculovirus infection. The level of inhibition was found to depend, both in duration and extent, on the concentration of injected RNAi. That relatively low levels of RNAi can inhibit protein synthesis driven by the strong polyhedrin (polh) promoter of AcMNPV, suggests that RNAi will find utility as an in vivo metabolic controller in metabolic engineering studies such as this one pertaining to protein expression.

Animals↗
PubMed2000

Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters.

The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.

Animals↗
PubMed2002

Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs.

The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3'-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3'-end of either strand, using 2'-O,4'-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3'-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3'-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3'-end is recognized by the RNA-induced silencing complex (RISC).

Animals↗
PubMed2003

Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference.

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.

Animals↗
PubMed2002

RNA interference of HIV replication.

Double-stranded RNA-mediated interference (RNAi) induces sequence-specific post-transcriptional gene silencing and has emerged as a powerful tool to silence gene expression in multiple organisms. In mammalian cells, duplexes of 21 nucleotide RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression. Recent research demonstrates the general use of siRNAs to specifically inhibit HIV-1 replication by targeting viral or cellular genes. Importantly, RNAi opens a new avenue for gene-based therapeutics.

Gene Silencing↗
PubMed2002

Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference.

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named "Dicer", functions in the RNA interference (RNAi) pathway by producing 21--23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse Chromosome (Chr) 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway.

Amino Acid Sequence↗
PubMed2003

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies.

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.

Amino Acid Sequence↗
PubMed2000

RNA interference for the organizer-specific gene Xlim-1 in Xenopus embryos.

Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in Xenopus embryos. First, expression of an exogenous luciferase gene as a reporter in embryos was reduced by coinjection with dsRNA corresponding to the luciferase gene. Next, injection of dsRNA for Xlim-1, a homeobox gene suggested to be involved in Spemann organizer functions, reduced the endogenous level of Xlim-1 mRNA and produced embryos with reduced eyes or anterior truncation at high efficiency. In addition, injection of an antisense expression construct of Xlim-1 elicited phenotypes very similar to those of Xlim-1 dsRNA-injected embryos. These results indicate the effectiveness of RNAi for loss of function studies in Xenopus embryos, and the importance of Xlim-1 in head formation.

Animals↗
PubMed2000

RNA interference: genetic wand and genetic watchdog.

In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi.

Animals↗
PubMed2002

RNA interference: advances and questions.

In animals and protozoa gene-specific double-stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post-transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasite Trypanosoma brucei represents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi in T. brucei and outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.

Animals↗
PubMed2003

Silencing the Drosophila ribosomal protein L14 gene using targeted RNA interference causes distinct somatic anomalies.

The Drosophila Minutes are haploinsufficient mutations that are defective in ribosomal protein (rp) production, resulting in short, thin bristles, delayed development and recessive lethality. In a Minute fly, the amount of rp gene messenger RNA (mRNA) is reduced to >or=50% of the normal amount of gene product, and becomes rate limiting for ribosome biogenesis, cell proliferation and growth. Haploinsufficiency increases the vulnerability to complete loss of gene function (homozygous null state) if hit by a second mutation. Because of the homozygous lethality, it has only been possible to study the effects of Minute mutations in heterozygous animals. To be able to study the consequences of a loss-of-function of an rp gene (0%>mRNA<50%) in developing and differentiated cells we used heritable RNA interference (RNAi) in combination with the yeast GAL4/UAS binary system to spatiotemporally knock down the ribosomal protein L14 (RpL14) gene. We show, at the RNA and phenotypic levels, that RNAi efficiently reduces RpL14 gene expression throughout development, causing lethality and distinct and dramatic somatic anomalies in both developing and differentiated cells.

Abnormalities, Multiple↗
PubMed2002

RNA interference reveals a requirement for myocyte enhancer factor 2A in activity-dependent neuronal survival.

RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system.

Animals↗
PubMed2003

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei.

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.

Animals↗
PubMed2003

Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi).

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.

Carcinoma↗