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At least 1,045 records · Page 58Linked to original sources
PubMed2000

A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains.

Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.

Adenosine Deaminase↗
PubMed2000

Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

Base Pairing↗
PubMed1999

A minor fraction of basic fibroblast growth factor mRNA is deaminated in Xenopus stage VI and matured oocytes.

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine in double-stranded regions of RNA. ADAR activity is in the nucleus in Xenopus laevis stage VI oocytes, and released into the cytoplasm at oocyte maturation. We previously demonstrated that a cytoplasmic double-stranded RNA (dsRNA) binding factor(s), cyto-dsRBP, protects microinjected dsRNA from the ADAR released at maturation. Here we describe experiments to determine whether an endogenous dsRNA, the duplex formed between sense and antisense transcripts of basic fibroblast growth factor (bFGF), is protected in a similar manner. Consistent with the presence of cyto-dsRBP, we observed that the majority of bFGF RNA was not deaminated, before or after maturation. However, a minor fraction of the bFGF RNA was deaminated whether the RNA was isolated from stage VI oocytes or matured oocytes. Since ADAR activity is in the nucleus in stage VI oocytes, our results suggest that a fraction of the bFGF RNAs are hybridized in the nucleus and are ADAR substrates. Adenosine deaminations result in A-to-G changes in cDNAs, so we quantified the fraction of modified molecules using restriction-enzyme assays of RT-PCR products. Caveats due to recombination during RT-PCR are discussed.

Adenosine Deaminase↗
PubMed1999

Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon.

Endogenous, 14 kb double-stranded RNAs (dsRNAs) have been found in two ecospecies of cultivated rice (temperate japonica rice and tropical japonica rice, Oryza sativa L.) and in wild rice (O. rufipogon, an ancestor of O. sativa). A comparison of the nucleotide and deduced amino acid sequences of the core regions of the RNA-dependent RNA polymerase domains found in these three dsRNAs suggested that these dsRNAs probably evolved independently within each host plant from a common ancestor. These dsRNAs were introduced into F1 hybrids by crossing cultivated rice and wild rice. Unusual cytoplasmic inheritance of these dsRNAs was observed in some F1 hybrids; the evolutionarily related dsRNAs were incompatible for each other, and the resident dsRNA of an egg cell from cultivated rice was excluded by the incoming dsRNA of a pollen cell from wild rice. Coexisting dsRNAs in the F1 hybrids segregated away from each other in the F2 plants. However, the total amount of these dsRNAs in the host cells remained constant (ca. 100 copies/cell). The stringent regulation of the dsRNA copy number may be responsible for their unusual inheritance.

Base Sequence↗
PubMed2002

The tomato RNA-directed RNA polymerase has no effect on gene silencing by RNA interference in transgenic mice.

Double-stranded RNA (dsRNA) has been shown to interfere with the function of specific genes in various invertebrate species. The application of dsRNA interference (RNAi) in vertebrates (zebrafish and mouse) is still limited to embryos and it is not clear whether the method is generally applicable. Using a transgenic mouse model we investigated whether a stably inherited dsRNA introduced as a transgene can interfere with the expression of a specific target gene in erythroid tissue during development. In our globin gene system we do not observe any specific RNA interference. We, therefore, also introduced another gene that may be involved in a mechanism of post transcriptional gene silencing (PTGS), namely RNA-dependent RNA polymerase (RdRP) that was proposed to be involved in producing RNAs that trigger PTGS in plants. However, even though the tomato RdRP is catalytically active in erythroid tissue, no RNAi was observed.

Animals↗
PubMed2002

Nucleotide sequences of double-stranded RNA segments from a hypovirulent strain of the white root rot fungus Rosellinia necatrix: possibility of the first member of the Reoviridae from fungus.

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41 x 10(6) to 2.95 x 10(6). Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5'-end (5'-ACAAUUU-3') and at the 3'-end (5'-UGCAGAC-3') were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.

3' Untranslated Regions↗
PubMed2003

Double-stranded RNA replicons associated with chloroplasts of a green alga, Bryopsis cinicola.

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4). The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [alpha-32P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.

Amino Acid Sequence↗
PubMed1996

A mammalian RNA editing enzyme.

Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

Adenosine Deaminase↗
PubMed2000

Heritable gene silencing in Drosophila using double-stranded RNA.

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

Animals↗
PubMed2003

Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi.

Transposable elements are stretches of DNA that can move and multiply within the genome of an organism. The Caenorhabditis elegans genome contains multiple Tc1 transposons that jump in somatic cells, but are silenced in the germ line. Many mutants that have lost this silencing have also lost the ability to execute RNA interference (RNAi), a process whereby genes are suppressed by exposure to homologous double-stranded RNA (dsRNA). Here we show how RNAi causes transposon silencing in the nematode germ line. We find evidence for transposon-derived dsRNAs, in particular to the terminal inverted repeats, and show that these RNAs may derive from read-through transcription of entire transposable elements. Small interfering RNAs of Tc1 were detected. When a germline-expressed reporter gene is fused to a stretch of Tc1 sequence, this transgene is silenced in a manner dependent on functional mutator genes (mut-7, mut-16 and pk732). These results indicate that RNAi surveillance is triggered by fortuitous read-through transcription of dispersed Tc1 copies, which can form dsRNA as a result of 'snap-back' of the terminal inverted repeats. RNAi mediated by this dsRNA silences transposase gene expression.

Alleles↗
PubMed2006

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

Animals↗
PubMed2004

Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

Active Transport, Cell Nucleus↗
PubMed2003

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines.

BACKGROUND: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. METHODS: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. RESULTS: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. CONCLUSIONS: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.

Antineoplastic Agents, Phytogenic↗
PubMed2006

The role of PACT in the RNA silencing pathway.

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.

Argonaute Proteins↗
PubMed2004

Inhibition of synapse assembly in mammalian muscle in vivo by RNA interference.

The formation of the vertebrate neuromuscular junction (NMJ) requires the receptor tyrosine kinase MuSK and the adaptor molecule rapsyn. Here, we report that the phenotypes of mice deficient in these two molecules can be reproduced by RNA interference (RNAi) in rat muscle in vivo. Specifically, double-stranded RNA (dsRNA) targeting MuSK and rapsyn inhibited the formation of the NMJ in rat muscle fibres in vivo, while dsRNA targeting nonessential proteins did not have any effect. Moreover, plasmids that trigger RNAi to MuSK induced the disassembly of existing NMJs. These results thus demonstrate for the first time the functionality of dsRNA in silencing endogenous genes in adult mammalian muscle in vivo. Moreover, they show that MuSK is also required for the maintenance of the NMJ, offering a mechanistic explanation for the myasthenia gravis caused by auto-antibodies to MuSK.

Animals↗
PubMed2002

Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene.

Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10-19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid co-injected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects.

Animals↗
PubMed2003

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.

Base Sequence↗
PubMed2001

Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).

During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.

Enzyme Activation↗