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PubMed2005

The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage.

Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways. dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I). Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA. These two pathways have previously been shown to be antagonistic. We show a novel interaction between components of the ADAR and RNAi pathways. Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi. Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I.U and U.I pairs. This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.

Adenosine Deaminase↗
PubMed2006

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system.

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

[Great potential of small RNAs: RNA interference and microRNA].

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

Humans↗
PubMed2002

Infectious bursal disease virus capsid protein VP3 interacts both with VP1, the RNA-dependent RNA polymerase, and with viral double-stranded RNA.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.

Animals↗
PubMed2005

Induction and mode of action of the viral stress-inducible murine proteins, P56 and P54.

Mammalian cells respond to virus infection or other viral stresses, such as double-stranded (ds) RNA and interferons (IFN), by robust and rapid induction of viral stress-inducible proteins. The induction and actions of one such protein, the human P56, have been extensively studied. However, little is known about the distantly related mouse proteins, MuP56 and MuP54. Here, we report that, in mouse cells, they could be induced by IFN, dsRNA or Sendai virus infection. MuP56 and MuP54 inhibited protein synthesis in vitro by binding to the "c", but not the "e", subunit of the translation initiation factor, eIF-3. The N-terminal region of the MuP54 was sufficient for inhibiting translation, but it and the corresponding region of MuP56 bound to two different regions of eIF3c. Thus, members of the human and murine P56 family have similar but non-identical functions.

Animals↗
PubMed1988

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity.

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Damaged DNA binding protein 1 in Drosophila defense reactions.

We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.

Animals↗
PubMed1995

Sequence of the M28 dsRNA: preprotoxin is processed to an alpha/beta heterodimeric protein toxin.

The killer and immunity phenotypes of K28 killer strains of Saccharomyces cerevisiae are determined by the 1.75-kb M28 dsRNA virus. In the plus strand, M28p, the K28 preprotoxin gene, comprises bases 13-1047 and is followed, after an additional 85 bases, by a 63-bp poly(A) sequence and a 553-base 3'-sequence. This 3'-sequence contains two potential stem-loop structures predicted to bind the L-A encoded cap-pol protein, initiating encapsidation; high-level expression results in curing of M1 dsRNA. Expression of M28p confers the complete K28 killer and immunity phenotype on a cell lacking M28 dsRNA. K28 toxin is a disulfide-bonded heterodimer of alpha (10.5 kDa) and beta (11 kDa) components whose N-termini correspond to M28p residues 50-61 and 246-257, respectively. alpha is preceded by a potentially redundant pair of secretion signal peptides; deletion of the first reduces toxin secretion by 75%. While M28p bears no sequence similarity to M1p, the K1 preprotoxin, the predicted patterns of processing by glycosylation and cleavage are remarkably similar. The beta N- and C-termini are probably processed by Kex2p and Kex1p, respectively; the mechanism of cleavage at the less typical sites bounding the alpha component is under investigation. While a kex2 delta mutation prevents toxin secretion, secreted toxin retains 20% activity in a kex1 delta mutant. Neither mutation affects immunity.

Amino Acid Sequence↗
PubMed2006

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

ATP-Binding Cassette Transporters↗
PubMed2004

The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome. We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates. This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA. Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site. By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.

Bacteriophage phi 6↗
PubMed2006

RNA interference machinery influences the nuclear organization of a chromatin insulator.

RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. We present evidence for a functional relationship between RNAi and the gypsy insulator of D. melanogaster. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression.

Animals↗
PubMed1988

Interferons-alpha/beta- and -gamma-resistant Friend cell variants exhibiting receptor sites for interferons but no induction of 2-5A synthetase and 67K protein kinase.

A number of Friend leukemia cell variants with a interferon-gamma (IFN-gamma)-resistant phenotype have been isolated. They appear resistant to the antiproliferative action of IFN-gamma and to the induction of the antiviral state assessed by Friend leukemia virus release and vesicular stomatitis virus yield. Selection was performed via a prolonged exposure to increasing amounts of highly purified recombinant IFN-gamma of wild-type Friend cells or of variant clones thereof already resistant to IFN-alpha/beta (Affabris et al., 1982, Virology 120, 441-452). Only the clones derived from IFN-alpha/beta-resistant variants showed a phenotype fully resistant to IFN-gamma treatment while keeping their previously acquired resistance to IFN-alpha/beta. These cells are not deficient in high-affinity receptors for IFN-gamma so that their resistant phenotype appears to be mediated by events distal to binding of IFN-gamma to its receptors. Furthermore, analysis of IFN-induced dsRNA-dependent 2-5A synthetase and 67K protein kinase enzymatic activities, biochemical markers for cellular responses to IFN, showed that both these activities were not induced in IFN-alpha/beta and IFN-gamma-resistant clones when treated with either type of IFN. Accordingly, no increased expression of 2-5A synthetase mRNA(s) could be detected by probing poly(A)+-enriched RNA from cells exposed to IFN-alpha/beta or IFN-gamma treatment with murine or human specific cDNAs. On the other hand, no major changes in restriction patterns of 2-5A synthetase gene(s) were observed in these variant cells by restriction endonuclease digestion and Southern blotting. In addition, analysis of 2-5A synthetase mRNA induction, performed on wild-type cells, showed that the kinetic of induction due to IFN-gamma treatment is slower than that obtained with IFN-alpha/beta.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Differential up-regulation of HLA class I molecules on neuronal and glial cell lines by virus infection correlates with differential induction of IFN-beta.

Adult neurons normally lack the expression of MHC class I molecules, which has implications on virus clearance from the central nervous system. The author previously demonstrated that HLA class I up-regulation in measles virus (MV)-infected glial cells is primarily mediated by IFN-beta. In contrast, this study demonstrates that MV-infection of the neuronal cell lines IMR-32 and CHP-126 fails to up-regulate HLA class I expression, which was associated with an inability of MV to induce IFN-beta in the neuronal cell lines. However, treatment with IFN-beta on coculture of the IMR-32 neuronal cell line with MV-infected glioma cells resulted in the up-regulation of HLA class I on the former, which could be neutralized by anti-IFN-beta Ab. The inability of MV to up-regulate HLA class I expression on the neuronal cell line IMR-32 was not virus specific because similar findings were observed with mumps virus or stimulation with the synthetic dsRNA polyinosinic polycytidylic acid (PIPC). Induction of IFN-beta gene expression by virus requires binding of NF-kappa B to the positive regulatory domain II element of the IFN-beta promoter. Our studies indicate that MV, TNF-alpha, or PIPC induces NF-kappa B (p50 and p65 subunits) binding to positive regulatory domain II in the glioma cell line. In contrast, such activity was induced by TNF-alpha but not MV or PIPC in the neuronal cell line IMR-32. This indicated that HLA class I expression is differentially regulated in glial and neuronal cell lines in response to MV, which correlates with differential binding of NF-kappa B to the IFN-beta promoter and induction of IFN-beta gene expression.

Base Sequence↗
PubMed1999

Double-stranded RNA-activated protein kinase (PKR) fused to green fluorescent protein induces apoptosis of human embryonic kidney cells: possible role in the Fas signaling pathway.

PKR is an interferon-inducible, double-stranded (ds) RNA-activated serine/threonine protein kinase, and has been shown to play roles in viral pathogenesis, cell growth and apoptosis. We expressed PKR as a fusion protein with enhanced jellyfish green fluorescence protein (EGFP) in human embryonic kidney 293 cells to visualize the effect of PKR transfection. The EGFP-fusion construct with wild-type PKR showed both auto- and substrate-phosphorylation activities independent of dsRNA, indicating EGFP-PKR is constitutively active. The EGFP-construct with a mutant PKR with the first RNA binding domain deleted still possessed kinase activities. On the other hand, the EGFP-fusion with a catalytically inactive mutant of PKR with the substitution of K at 296 with R, which has been shown to have tumorigenic properties, did not possess kinase activities. Transfection of the constitutive active forms of EGFP-PKR constructs induced apoptosis in 293 cells without dsRNA, whereas the EGFP-fusion with the catalytically inactive mutant did not cause apoptosis but rather protected cells from Fas-induced cell death. In addition, Fas-stimulation increased endogenous PKR activities. These results constitute evidence that PKR is sufficient to induce apoptosis, and plays a role in Fas-mediated apoptosis.

Animals↗
PubMed2004

PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation.

During the complex life cycle of Plasmodium falciparum, divided between mosquito and human hosts, the regulation of morphologic changes implies a fine control of transcriptional regulation. Transcriptional control, however, and in particular its molecular actors, transcription factors and regulatory motifs, are as yet poorly described in Plasmodium. In order to decipher the molecular mechanisms implicated in transcriptional regulation, a transcription factor belonging to the tryptophan cluster family was studied. In a previous work, the PfMyb1 protein, contained in nuclear extracts, was shown to have DNA binding activity and to interact specifically with myb regulatory elements. We used long pfmyb1 double-stranded RNA (dsRNA) to interfere with the cognate messenger expression. Parasite cultures treated with pfmyb1 dsRNA exhibited a 40% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA, and parasite mortality occurred during trophozoite to schizont transition. In addition, the pfmyb1 transcript and protein decreased by as much as 80% in treated trophozoite cultures at the time of their maximum expression. The global effect of this partial loss of transcript and protein was investigated using a thematic DNA microarray encompassing genes involved in signal transduction, cell cycle and transcriptional regulation. SAM software enabled us to identify several genes that were differentially expressed and probably directly or indirectly under the control of PfMyb1. Using chromatin immuno-precipitation, we demonstrated that PfMyb1 binds, within the parasite nuclei, to several promoters and therefore participates directly in the transcriptional regulation of the corresponding genes. This study provides the first evidence of a regulation network involving a Plasmodium transcription factor.

Animals↗
PubMed2006

Zinc ions stimulate the cooperative RNA binding of hordeiviral gammab protein.

A small regulatory gammab protein of the Poa semilatent hordeivirus (PSLV) contains two zinc finger-like motifs separated by a basic motif in the N-terminal part and a C-terminal coiled-coil motif. Interactions of the recombinant PSLV gammab protein and its mutants with various RNAs (ssRNA, dsRNA, ssRNA oligonucleotides) and ssDNA were studied in gel-shift assays. The results demonstrated that zinc ions are essential for effective nucleic-acid-binding activity of the gammab protein, suggesting the important role of zinc finger motifs in these interactions. Deletion of the C-proximal coiled-coil region did not affect highly cooperative RNA-protein binding, indicating that the N-terminal part of the protein contributes to the protein-protein interactions needed for the protein-RNA cooperativity.

Amino Acid Sequence↗
PubMed2003

Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis.

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin we have depleted DmSMC4, a subunit of the complex, from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. Our results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution.

Adenosine Triphosphatases↗
PubMed2006

Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex.

The Drosha-DGCR8 complex initiates microRNA maturation by precise cleavage of the stem loops that are embedded in primary transcripts (pri-miRNAs). Here we propose a model for this process that is based upon evidence from both computational and biochemical analyses. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction. Purified DGCR8, but not Drosha, interacts with pri-miRNAs both directly and specifically, and the flanking ssRNA segments are vital for this binding to occur. Thus, DGCR8 may function as the molecular anchor that measures the distance from the dsRNA-ssRNA junction. Our current study thus facilitates the prediction of novel microRNAs and will assist in the rational design of small hairpin RNAs for RNA interference.

Animals↗