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PubMed2004

Characteristics of RNA silencing in plants: similarities and differences across kingdoms.

RNA silencing is a collective term that encompasses the sequence of events that leads to the targeted degradation of cellular mRNA and thus to the silencing of corresponding gene expression. RNA silencing is initiated after introduction into the host genome of a gene that is homologous to an endogenous gene. Transcription of the introduced gene results in the formation of double-stranded RNA (dsRNA) that is cut into smaller dsRNA species termed small interfering RNAs (siRNAs) by an RNaseIII-like enzyme called 'Dicer'. siRNAs associate with a protein complex termed the 'RNA-induced silencing complex' (RISC), which mediates the binding of one strand of siRNAs with mRNAs transcribed from the native 'target' gene. The binding of siRNAs with native gene mRNAs earmarks native gene mRNAs for destruction, resulting in gene silencing. In plants, RNA silencing appears to serve as a defence mechanism against viral pathogens and also to suppress the activity of virus-like mobile genetic elements. In an apparent response to RNA silencing, some plant viruses express suppressors of RNA silencing. RNA silencing also is directly implicated in the regulation of the function(s) of microRNAs, which are the key determinants in an additional cellular mechanism related to the translational repression of genes, the effect of which ultimately impinges on development. The high degree of sequence similarity that exists between genes involved in RNA silencing in widely different organisms underscores the conserved nature of many aspects of the RNA silencing mechanism. However, depending (for example) on the precise nature of the target gene involved, there also are significant differences in the silencing pathways that are engaged by various organisms.

Gene Expression Regulation, Plant↗
PubMed2006

Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance.

The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed (15)N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.

Amino Acid Sequence↗
PubMed2003

Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia.

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.

Apoptosis↗
PubMed2005

Isolation of white gene orthologue of the sawfly, Athalia rosae (Hymenoptera) and its functional analysis using RNA interference.

We isolated and characterized the white gene orthologue of the sawfly, Athalia rosae (Hymenoptera). The A. rosae white (Ar white) cDNA cloned was 2058-bp long encoding 685 amino acids in a single open reading frame (ORF). Comparison of the cDNA sequence with the genomic DNA sequence revealed that the ORF was derived from 11 exons. Ar white was a single copy gene as evidenced by genomic Southern blotting and its cytological localization on the metaphase chromosomes. The deduced amino acid sequence aligned well with known insect white orthologous gene products sharing conserved regions such as the ATP-binding motif and the six transmembrane-spanning segments. Expression of Ar white was detected at embryonic and pupal stages by Northern blotting. In situ hybridization detected the embryonic expression in a pair of the lateral tips of protocephalic placodes from where optic organs are formed. Ar white function was examined using double-stranded RNA (dsRNA)-mediated interference. The synthesized dsRNA targeting Ar white transcripts caused a decrease in the level of the original mRNAs, and resulted in the white phenocopy in the embryonic eye pigmentation when microinjected into eggs from wild-type females. The effects occurred in a dose-dependent manner.

ATP-Binding Cassette Transporters↗
PubMed2000

RNA recognition by a Staufen double-stranded RNA-binding domain.

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

Amino Acid Sequence↗
PubMed1993

Investigation of a killer strain of Zygosaccharomyces bailii.

The yeast Zygosaccharomyces bailii strain 412 was found to liberate a killer toxin (KT412) lethal to sensitive strains of Saccharomyces cerevisiae and Candida glabrata. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular protein was purified by gel filtration and ion-exchange chromatography. Gel filtration and SDS-PAGE of the electrophoretically homogeneous killer protein indicated an apparent molecular mass of 10 kDa. The killer toxin KT412 is probably not glycosylated since it did not show any detectable carbohydrate structures. KT412 was bound to sensitive but not to resistant yeast cells. The mannan, and not the glucan, fraction of the cell wall of the sensitive yeast was the primary target for the killer toxin binding. The killer strain Z. bailii 412 contained three double-stranded RNA plasmids of 1.9, 2.9 and 4.0 kb. Curing by cycloheximide resulted in the concomitant loss of killer activity and the 1.9 kb dsRNA species that is therefore regarded as equivalent to the killer-toxin-coding M-plasmids of S. cerevisiae.

Cell Wall↗
PubMed1994

The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.

The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.

Amino Acid Sequence↗
PubMed2007

Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

Animals↗
PubMed2006

Are viral-encoded microRNAs mediating latent HIV-1 infection?

The Human Immunodeficiency Virus type 1 (HIV-1), a member of the lentivirus subfamily, infects both dividing and nondividing cells and, following reverse transcription of the viral RNA genome, integrates into the host chromatin where it enters into a latent state. Many of the factors governing viral latency remain unresolved and current antiviral treatment regimens are largely ineffective at eliminating cellular reservoirs of latent virus. The recent identification of microRNA (miRNA) encoding sequences embedded in the HIV-1 genome, and the discovery of functional virus-derived miRNAs, suggests a role for RNA Interference (RNAi) in the regulation of HIV-1 gene expression. Recently, the mammalian RNAi machinery was shown to regulate gene expression epigenetically by transcriptional modulation, providing a direct link between RNAi and a mechanism for inducing latency. Interestingly, both HIV-1 Tat, and the host TAR RNA-binding protein (TRBP), bind to the transactivating response (TAR) RNA of HIV-1 and affect the function of RNAi in human cells. Specifically, TRBP, a cofactor in Tat-TAR interactions, is a vital component of Dicer-mediated dsRNA processing. These novel observations support a central role for HIV-1 and associated host factors in regulating cellular RNAi and viral gene expression through RNA directed processes. Thus, HIV-1 may have evolved mechanisms to exploit the RNAi pathway at both the transcriptional and posttranscriptional level to affect and/or maintain a latent infection.

Gene Expression Regulation, Viral↗
PubMed1992

Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae.

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.

Base Sequence↗
PubMed2001

Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage.

It is now evident that members of the RNase III family of nucleases have central roles in prokaryotic and eukaryotic RNA maturation and decay pathways. Ongoing research is uncovering new roles for RNase III homologs. For example, the phenomena of RNA interference (RNAi) and posttranscriptional gene silencing (PTGS) involve dsRNA processing, carried out by an RNase III homolog. We anticipate an increased focus on the mechanism, regulation, and biological roles of RNase III orthologs. Although the differences in the physicochemical properties of RNase III orthologs, and distinct substrate reactivity epitopes and ionic requirements for optimal activity, may mean that the protocols describe here are not strictly transferrable, the affinity purification methodology, and substrate preparation and use should be generally applicable.

Base Sequence↗
PubMed1994

IFN consensus sequence binding protein (ICSBP) is a conditional repressor of IFN inducible promoters.

IFN stimulated genes (ISGs) contain common DNA motif termed IFN consensus sequence (ICS) at their promoters that enable IFN responsiveness. Different transcription factors capable of interacting with the ICS have been described. Previously, we reported the cloning of a factor capable of binding to the ICS (ICSBP) that demonstrates similarity at DNA the binding domain with three other ICS binding factor, i.e. IRF-1, IRF-2 and ISGF3 gamma. ICSBP is expressed constitutively in hematopoietic cells and its expression is further induced by IFN-gamma. This is a negative trans-acting regulator of ISGs; however, its effect is attenuated following prolonged exposures of cells to both types of IFNs. In this communication, we show that short exposures of cells to IFNs (priming) are sufficient to alleviate ICSBP mediated repression. Further, exposure of primed cells to the synthetic dsRNA (polyl-polyC) results in total abrogation of ICSBP repression. In an attempt to unravel the molecular mechanism governing this conditional repression of ICSBP, the direct involvement of transcriptional activator IRF-1 is demonstrated. We postulate that constitutive expression of ICSBP in hematopoietic cells is mediating submaximal expression of ISGs such as MHC class I. Our data demonstrate that IRF-1 competes with ICSBP for the binding to the ISRE element, resulting in the alleviation of ICSBP repression. Thus, the magnitude of ISGs expression is a result of a fine balance between positive and negative regulators.

Antisense Elements (Genetics)↗
PubMed2005

Cleavage of dsRNAs hyper-edited by ADARs occurs at preferred editing sites.

Long double-stranded RNAs (dsRNAs) may undergo covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), whereby up to 50-60% of adenosine residues are converted to inosine. Previously, we have described a ribonuclease activity in various cell extracts that specifically targets dsRNAs hyper-edited by ADARs. Such a ribonuclease may play an important role in viral defense, or may alternatively be involved in down-regulation of other RNA duplexes. Cleavage of hyper-edited dsRNA occurs within sequences containing multiple IU pairs but not in duplexes that contain either isosteric GU pairs or Watson-Crick base pairs. Here, we describe experiments aimed at further characterizing cleavage of hyper-edited dsRNA. Using various inosine-containing dsRNAs we show that cleavage occurs preferentially at a site containing both IU and UI pairs, and that inclusion of even a single GU pair inhibits cleavage. We also show that cleavage occurs on both strands within a single dsRNA molecule and requires a 2'-OH group. Strikingly, we show that ADAR1, ADAR2 or dADAR all preferentially generate the preferred cleavage site when hyper-editing a long dsRNA.

Adenosine Deaminase↗
PubMed2004

The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter.

Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN-beta promoter in reporter gene assays and significantly enhanced the activation of the IFN-beta promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN-beta promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN-beta promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.

Animals↗
PubMed2006

Single-stranded RNA viruses inactivate the transcriptional activity of p53 but induce NOXA-dependent apoptosis via post-translational modifications of IRF-1, IRF-3 and CREB.

To characterize the mechanisms underlying apoptosis induced by viral infection, transcriptional activation of genes encoding members of the 'BH3-only' family of proteins was analysed during the course of virus infection. Among these genes, only NOXA is transcriptionally activated by vesicular stomatitis virus (VSV), sendai virus (SV), measles virus, herpes simplex virus, or dsRNA and required for efficient apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53, but requires the presence of interferon regulatory factor 1 (IRF-1), IRF-3 and cAMP-responsive element binding protein (CREB). Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving different pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by Toll like receptor 3 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection correlates with a 10 kDa increase in its molecular weight, suggesting a covalent linkage with a previously unknown regulatory polypeptide.

Apoptosis↗
PubMed1989

Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase.

Interferon treatment of mammalian cells induces a double-stranded (ds) RNA-dependent protein kinase known as DAI. When activated, DAI phosphorylates the alpha-subunit of eukaryotic initiation factor eIF-2, impairing its ability to be recycled and leading to the inhibition of protein synthesis. We have identified a novel DAI substrate in the ribosomal salt wash of rabbit reticulocyte lysates. This substrate is a 90-kDa polypeptide which has been purified to apparent homogeneity. It can be cross-linked by ultraviolet irradiation to adenovirus VA RNAI, a small RNA polymerase III transcript RNA which acts as an inhibitor of DAI. As assayed by a nitrocellulose filter binding assay, the 90-kDa polypeptide is also able to associate with authentic double-stranded RNA, but not single-stranded RNA, made in vitro. Thus, this newly identified substrate of DAI appears to have affinity for dsRNA structures and may be involved in dsRNA-regulated processes in the reticulocyte. Polyclonal and monoclonal antibodies directed against the 90-kDa polypeptide co-precipitate DAI, suggesting that these two proteins may exist as a complex.

Adenoviruses, Human↗
PubMed2000

Cloning of an Mx cDNA from Atlantic halibut (Hippoglossus hippoglossus) and characterization of Mx mRNA expression in response to double-stranded RNA or infectious pancreatic necrosis virus.

Mx proteins are GTPases that are specifically induced by type I interferons (IFN) in vertebrates. Some mammalian Mx proteins have antiviral activity against certain RNA viruses. A 2.3-kb full-length cDNA clone of an Atlantic halibut Mx gene was isolated from a liver cDNA library. The open reading frame (ORF) predicts a 622 amino acid protein of 71.2 kDa possessing a tripartite GTP binding motif, a dynamin signature, and a leucine zipper motif, which are conserved in all known Mx proteins. The C-terminal half contains a putative bipartite nuclear localization signal. The deduced halibut Mx protein showed approximately 76% sequence identity with the Atlantic salmon and rainbow trout Mx proteins, 55% identity with the human MxA, and 48% identity with the chicken Mx protein. Based on sequence comparison of 554-bp Mx cDNA fragments, the Atlantic halibut Mx showed more relationship with the perch and turbot than the salmonid Mx genes. Halibut appears to possess at least two Mx loci, as suggested by Southern blot analysis of genomic DNA. Two halibut Mx transcripts (2.2 kb and 2.6 kb) were strongly induced in vivo by the double-stranded RNA (dsRNA) poly I:C or infectious pancreatic necrosis virus (IPNV) in all organs studied.

Amino Acid Sequence↗
PubMed1994

Characterization of an antisense Inr element in the eIF-2 alpha gene.

We recently discovered an opposing initiator promoter (Inr) downstream of the sense promoter region of the eIF-2 alpha gene (Silverman, T., Noguchi, M., and Safer, B. (1992) J. Biol. Chem. 267, 9738-9742). By reverse transcriptase/polymerase chain reaction analysis of G0 and activated (G1) T-lymphocyte RNAs, overlapping sense and antisense transcripts are now identified. Sense transcription of the eIF-2 alpha gene proceeds from left to right to generate alpha-mRNA; antisense transcription proceeds from right to left to generate RNA, having a sequence complementary to eIF-2 alpha mRNA. Upstream indicates a position 5' relative to the transcription start site. Using DNase I footprint analysis and EMSA, we have found a potential cis-regulatory sequence immediately upstream of the Inr which binds a 43-kDa protein. In addition to conferring protection against DNase I (+457 to +474), the factor also generates hypersensitive sites directly over the Inr (+447 to +457). Insertion of the Inr footprint region into a luciferase reporter gene construct increases expression 150-fold. While mutation of the Inr conserved sequence decreases luciferase activity by 50%, mutation of the 43-kDa factor binding site inhibits luciferase activity by 20%. Sense orientation of the Inr footprint region decreases activity by 80%. The 43-kDa Inr-associated binding protein may be involved in allowing access of RNA polymerase II transcription complexes ot the initiation site of this TATA-less gene. A model for the regulation of eIF-2 alpha expression involving the rapid degradation of dsRNA generated by the relative activities of the two overlapping and opposing promoters is proposed.

Base Sequence↗