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PubMed2006

RNA delivery by heat shock protein-70 into mammalian cells: a preliminary study.

Heat shock proteins (Hsps) are ubiquitous molecular chaperones with indispensable roles in assisting protein folding and giving protection from proteotoxic environmental harm. Members of the 70-kDa heat shock protein family have been demonstrated to recognize and bind with distinguished RNA sequences, which function as determinants of eukaryotic mRNA stability. We have earlier identified the molecular domains involved in RNA-binding and characterized in detail the specificity, affinity and some regulatory aspects of this molecular interaction using various deletion mutants and homologues of Hsp70. We have shown that wild type, but not any of the tested truncated mutants of Hsp70, is efficiently taken up by P388 mouse macrophage cells. Here we addressed the question of whether Hsp70 is capable of delivering bound RNA into mammalian cells. Employing fluorescence and confocal microscopy, we demonstrated that full length Hsp70 facilitates the uptake of RNA molecules into the cytoplasm of mammalian cells. We propose that further optimization of this system might enable the development of a valuable tool to deliver RNA molecules, such as siRNA, dsRNA or other regulatory RNA sequences to probe or influence various regulatory processes in eukaryotic cells.

Animals↗
PubMed1995

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Base Sequence↗
PubMed1991

A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.

Amino Acid Sequence↗
PubMed1995

Atomic force microscopy of nucleoprotein complexes.

Recent data on the AFM studies of nucleoprotein complexes of different types are reviewed in this paper. The first section describes the progress in the sample preparation methods for AFM studies of nucleic acids and nucleoprotein complexes. The second part of this paper reviews AFM data on studies of complexes of DNA with regulatory proteins. These studies include two different types of DNA distortion induced by proteins binding: local bending of DNA at sites of protein binding and formation of large loops due to protein-protein interactions between molecules bound to distant sites along the DNA molecules (DNA looping). The prospects for use of AFM for physical mapping of genomes are discussed in this section as well. The third part of the paper reviews data on studies of complexes of DNA with non-sequence specific binding proteins. Special emphasis is given to studies of chromatin which have resulted in progress in the understanding of structure of native chromatin fiber. In this section, novel data on AFM studies of RecA-DNA filaments and complexes of dsRNA with the dsRNA-specific protein p25 are also presented. Discussion of the substrate preparation procedures in relation to the AFM studies of nucleoprotein complexes is given in the final section.

Animals↗
PubMed2003

Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains.

PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor 3 (TLR3), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR3 expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR3 was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (Interferon-alpha/beta receptor) suppressed MV-induced TLR3 induction, indicating that type I IFN, IFN-alpha/beta, is critical for MV-mediated TLR3 induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR3 expression. A virus-responsive element that up-regulates TLR3 was identified in the TLR3-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -30bp of TLR3 promoter responded to MV to induce TLR3 expression. This further indicates the importance of type I IFN for TLR3 up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR3 had been induced beforehand. Thus, the MV-induced expression of TLR3 may reflect amplified IFN production that plays a part in host defense to viral infection.

Cell Line↗
PubMed2001

Involvement of proteasomes in gene induction by interferon and double-stranded RNA.

Cytokine induced gene expression is mediated through the ligand-dependent activation of the janus kinase (jak)/signal transducer and activator of transcription (STAT) signal transduction pathway. The ubiquitin proteasome pathway functions in the controlled degradation of cellular proteins, and regulates cytokine signal transduction through the degradation of specific signaling components. Interferon (IFN) treatment induces genes that function in ubiquitin conjugation, suggesting a reciprocal regulation of proteasome activity and IFN action; however, a role for the proteasome in IFN-alpha-induced gene expression has not been examined. In this report, we find that proteasome inhibitors markedly reduce the induction of interferon-stimulated-gene 15 (ISG15), ISG43, and STAT1 by IFN-alpha and double-stranded RNA (dsRNA). The reduction in gene expression by proteasome inhibitors was dose-dependent, and was specific for ISGs. Neither STAT1 phosphorylation nor ISGF-3 activation was affected by proteasome inhibition at early times post-IFN treatment. Cycloheximide treatment diminished the effect of proteasome inhibitors on ISG induction, implicating an IFN/dsRNA-induced protein in this activity. These findings demonstrate that a functional proteasome is required for optimal ISG induction, and are consistent with a model in which IFN and dsRNA induce a proteasome-sensitive repressor of ISG expression.

Animals↗
PubMed2004

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation.

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.

Cell Line↗
PubMed2004

Novel predicted peptidases with a potential role in the ubiquitin signaling pathway.

A multi-pronged strategy including extensive sequence searches, structural modeling, and analysis of contextual information extracted from domain architectures, genetic screens, and large-scale protein-protein interaction analyses was employed to predict previously undetected components of the eukaryotic ubiquitin (Ub) signaling system. Two novel groups of proteins that are likely to function as de-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The first group of putative DUBs, designated PPPDE superfamily (after Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidases with a circularly permuted papain-like fold. The inference of the likely DUB function of the PPPDE superfamily proteins is based on the fusions of the catalytic domain to Ub-binding PUG (PUB)/UBA domains and a novel alpha-helical Ub-associated domain (the PUL domain, after PLAP, Ufd3p and Lub1p). The presence of the PPPDE superfamily proteins in most eukaryotic lineages, including basal ones, such as Giardia, suggests a role in deubiquitination of highly conserved proteins involved in key cellular functions, such as cell cycle control. In addition to eukaryotic proteins, the PPPDE superfamily includes predicted proteases from several groups of double-stranded RNA viruses and one single-stranded DNA virus. The apparent recruitment of DUBs for viral polyprotein processing seems to represent a common theme in evolution of viruses. The second group of putative DUBs identified in this study is the WLM (Wss1p-like metalloproteases) family of the Zincin-like superfamily of Zn-dependent peptidases, which are linked to the Ub-system by virtue of fusions with the UB-binding PUG (PUB), Ub-like, and Little Finger domains. More specifically, genetic evidence implicates the WLM family in de-SUMOylation. If validated experimentally, the WLM family proteins will represent the first case of a Zincin-like metalloprotease involvement in Ub-signaling.

Amino Acid Sequence↗
PubMed2001

RNA interference is mediated by 21- and 22-nucleotide RNAs.

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

Animals↗
PubMed2004

Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation.

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

Adenosine Triphosphatases↗
PubMed2006

The role of IFN-alpha and nitric oxide in the release of HMGB1 by RAW 264.7 cells stimulated with polyinosinic-polycytidylic acid or lipopolysaccharide.

High mobility group protein 1 (HMGB1) is a nonhistone nuclear protein with a dual function. Inside the cell, HMGB1 binds to DNA and modulates a variety of processes, including transcription. Outside the cell, HMGB1 displays cytokine activity and can promote inflammation, serving as a mediator in models of shock and arthritis. In in vitro studies, proinflammatory molecules such as LPS, lipoteichoic acid, dsRNA, TNF-alpha, and IFN-gamma can induce HMGB1 release from macrophages. To define further the release process, we investigated the role of the downstream mediators, NO and IFN-alpha, in the release of HMGB1 from RAW 264.7 macrophage cells stimulated with LPS or polyinosinic-polycytidylic acid (poly(I:C)). In these experiments, 1400W, an inhibitor of NO production by the inducible NO synthase, reduced HMGB1 release stimulated by LPS, but not poly(I:C), whereas neutralizing IFN-alpha prevented HMGB1 release induced by poly(I:C), but not LPS. The addition of an NO donor and rIFN-alpha to RAW 264.7 cells caused HMGB1 release. Furthermore, inhibition of JNK activation attenuated HMGB1 release induced by either LPS or poly(I:C). Analysis of bone marrow-derived macrophages stimulated by LPS or poly(I:C) showed patterns of HMGB1 release similar to those of RAW 264.7 cells. Together, these experiments indicate that, although both LPS and poly(I:C) induce HMGB1 release from RAW 264.7 cells and murine macrophages, the response is differentially dependent on NO and IFN-alpha.

Animals↗
PubMed2002

The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis.

BACKGROUND: The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. RESULTS: The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes. First, the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation. Second, the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. CONCLUSIONS: Members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes.

Animals↗
PubMed2000

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila.

BACKGROUND: The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. RESULTS: When expressed under the control of a heat-inducible promoter, dsRNA interfered efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generated defects in larval molting and metamorphosis, resulting in animals that failed to pupariate or prepupae that died with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor had no effect on puparium formation, but led to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that expressed either EcR or betaFTZ-F1 dsRNA showed defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. CONCLUSIONS: These results confirm and extend our understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila.

Animals↗
PubMed2002

The role of RNA editing by ADARs in RNAi.

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.

Adenosine Deaminase↗
PubMed2006

Promoters of type I interferon genes from Atlantic salmon contain two main regulatory regions.

Recognition of viral nucleic acids by vertebrate host cells results in the synthesis and secretion of type I interferons (IFN-alpha/beta), which induce an antiviral state in neighboring cells. We have cloned the genes and promoters of two type I IFNs from Atlantic salmon. Both genes have the potential to encode IFN transcripts with either a short or a long 5'-untranslated region, apparently controlled by two distinct promoter regions, PR-I and PR-II, respectively. PR-I is located within 116 nucleotides upstream of the short transcript and contains a TATA-box, two interferon regulatory factor (IRF)-binding motifs, and a putative nuclear factor kappa B (NFkappaB)-binding motif. PR-II is located 469-677 nucleotides upstream of the short transcript and contains three or four IRF-binding motifs and a putative ATF-2/c-Jun element. Complete and truncated versions of the promoters were cloned in front of a luciferase reporter gene and analyzed for promoter activity in salmonid cells. Constructs containing PR-I were highly induced after treatment with the dsRNA poly(I:C), and promoter activity appeared to be dependent on NFkappaB. In contrast, constructs containing exclusively PR-II showed poor poly(I:C)-inducible activity. PR-I is thus the main control region for IFN-alpha/beta synthesis in salmon. Two pathogenic RNA viruses, infectious pancreatic necrosis virus and infectious salmon anemia virus, were tested for their ability to stimulate the minimal PR-I, but only the latter was able to induce promoter activity. The established IFN promoter-luciferase assay will be useful in studies of host-virus interactions in Atlantic salmon, as many viruses are known to encode proteins that prevent IFN synthesis by inhibition of promoter activation.

Animals↗
PubMed1991

Substrate specificity of the dsRNA unwinding/modifying activity.

Double-stranded RNA (dsRNA) unwinding/modifying activity, which is present in a wide range of eukaryotic cells, has been previously shown to convert up to 50% of adenosine residues to inosines within intermolecular dsRNA. In the present study, we report that this activity also modifies, though slightly less efficiently, intramolecular double-stranded regions of synthetic RNAs. Our results widen the range of the possible biological substrates for the activity since many stem and loop type RNA secondary structures (intramolecular dsRNA), present in eukaryotic as well as viral transcripts, can potentially serve as substrates. In addition, we have found that the dsRNA unwinding/modifying activity requires a double-stranded region of at least 15-20 base pairs (bp) for substrate recognition. Furthermore, modification efficiency was found to be critically dependent on the length of the double-stranded region; as the size decreased below 100 bp, it dropped precipitously. Our results suggest that efficient modification may occur only with relatively long (greater than 100 bp) dsRNA, perhaps because multiple copies of the enzyme must be bound.

Base Sequence↗
PubMed1998

RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans.

Introduction of exogenous double-stranded RNA (dsRNA) into Caenorhabditis elegans has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. In this study we present evidence that the primary interference effects of dsRNA are post-transcriptional. First, we examined the primary DNA sequence after dsRNA-mediated interference and found no evidence for alterations. Second, we found that dsRNA-mediated interference with the upstream gene in a polar operon had no effect on the activity of the downstream gene; this finding argues against an effect on initiation or elongation of transcription. Third, we observed by in situ hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the target for interference and suggest a mechanism that degrades the targeted RNA before translation can occur. This mechanism is not dependent on the SMG system, an mRNA surveillance system in C. elegans responsible for targeting and destroying aberrant messages. We suggest a model of how dsRNA might function in a catalytic mechanism to target homologous mRNAs for degradation.

Animals↗
PubMed1992

Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.

The double-stranded RNA-dependent protein kinase (dsRNA-PK) is thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs). Studies examining the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Autophosphorylation and consequent activation of dsRNA-PK in vitro and in vivo result in phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) and inhibition of protein synthesis. Expression of a functionally defective mutant of human dsRNA-PK in NIH 3T3 cells resulted in malignant transformation, suggesting that dsRNA-PK may function as a suppressor of cell proliferation and tumorigenesis.

Amino Acid Sequence↗