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At least 757 records · Page 42Linked to original sources
PubMed2006

Functional replacement of Trypanosoma brucei Argonaute by the human slicer Argonaute2.

RNA interference (RNAi) is widespread throughout the eukaryotic lineage, from protozoa to man. Central to all RNAi phenomena is a member of the Argonaute protein family, and, in the case of dsRNA-triggered mRNA cleavage, the Ago protein functions as the RNAi endonuclease or slicer. However, at present there is no definite experimental evidence that slicer Argonautes can be interchanged between distantly related organisms. Here, we show that the human slicer Argonaute2 (HsAgo2), but not HsAgo1, functions in RNAi in the early divergent protozoan Trypanosoma brucei, thus mimicking the situation in mammalian cells. This finding indicates that the basic features of the RNAi mechanism are conserved from T. brucei to man.

Animals↗
PubMed2005

Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila.

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

Active Transport, Cell Nucleus↗
PubMed2005

Post-transcriptional gene silencing by siRNAs and miRNAs.

Recent years have seen a rapid increase in our understanding of how double-stranded RNA (dsRNA) and 21- to 25-nucleotide small RNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), control gene expression in eukaryotes. This RNA-mediated regulation generally results in sequence-specific inhibition of gene expression; this can occur at levels as different as chromatin modification and silencing, translational repression and mRNA degradation. Many details of the biogenesis and function of miRNAs and siRNAs, and of the effector complexes with which they associate have been elucidated. The first structural information on protein components of the RNA interference (RNAi) and miRNA machineries is emerging, and provides some insight into the mechanism of RNA-silencing reactions.

Animals↗
PubMed1998

Type I interferons are essential mediators of apoptotic death in virally infected cells.

BACKGROUND: The interferons (IFNs) have been extensively studied in the context of host defence against viral infection. In the established model of IFN action, virally infected cells secrete type I IFNs (IFN-alpha/beta) which induce an antiviral state in uninfected cells. However, it is not clear how IFNs function on the infected cells. It has been reported that cells infected by some viruses die by apoptosis. RESULTS: In the present study, we found that three types of viruses commonly induce apoptosis in primary cell cultures. Importantly, we observed that virus-induced apoptosis was inhibited by anti-IFN-alpha/beta antibodies, and in cells lacking either the type I IFN receptor 1 (IFNAR1) or its downstream mediator, Stat1 (Signal transducer and activator of transcription 1). IFN-alpha treatment by itself did not induce apoptosis unless it was combined with transfection by double-stranded RNA (dsRNA), which is normally generated during the course of viral infection. CONCLUSION: These results indicate a novel antiviral function of the type I IFNs, i.e. the selective induction of apoptosis in virally infected cells. In effect, these IFNs have a bifunctional role in limiting the spread of virus; eliciting an antiviral state in uninfected cells while promoting apoptosis in infected cells. Our results may help explain why IFNs are sometimes useful in the treatment of viral diseases and will provide further insight into the mechanisms of virus-induced pathogenesis.

Animals↗
PubMed2005

Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids.

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. We show that CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. We analysed the consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, we discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes were still able to compact, paving the way for the identification of further components or activities required for this essential process.

Adenosine Triphosphatases↗
PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗
PubMed2004

A small modulatory dsRNA specifies the fate of adult neural stem cells.

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels.

Animals↗
PubMed2006

Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways.

In plants, animals, and fungi, members of the Dicer family of RNase III-related enzymes process double-stranded RNA (dsRNA) to initiate small-RNA-mediated gene-silencing mechanisms. To learn how C. elegans Dicer, DCR-1, functions in multiple distinct silencing mechanisms, we used a mass-spectrometry-based proteomics approach to identify DCR-1-interacting proteins. We then generated and characterized deletion alleles for the corresponding genes. The interactors are required for production of three species of small RNA, including (1) small interfering RNAs (siRNAs), derived from exogenous dsRNA triggers (exo-siRNAs); (2) siRNAs derived from endogenous triggers (endo-siRNAs); and (3) developmental regulatory microRNAs (miRNAs). One interactor, the conserved RNA-phosphatase homolog PIR-1, is required for the processing of a putative amplified DCR-1 substrate. Interactors required for endo-siRNA production include ERI-1 and RRF-3, whose loss of function enhances RNAi. Our findings provide a first glimpse at the complex biochemical niche of Dicer and suggest that competition exists between DCR-1-mediated small-RNA pathways.

Amino Acid Sequence↗
PubMed1999

RAX, a cellular activator for double-stranded RNA-dependent protein kinase during stress signaling.

The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor-2. PKR is activated by viral induced dsRNA and thought to be involved in the host antiviral defense mechanism. PKR is also activated by various nonviral stresses such as growth factor deprivation, although the mechanism is unknown. By screening a mouse cDNA expression library, we have identified an ubiquitously expressed PKR-associated protein, RAX. RAX has a high sequence homology to human PACT, which activates PKR in the absence of dsRNA. Although RAX also can directly activate PKR in vitro, overexpression of RAX does not induce PKR activation or inhibit growth of interleukin-3 (IL-3)-dependent cells in the presence of IL-3. However, IL-3 deprivation as well as diverse cell stress treatments including arsenite, thapsigargin, and H2O2, which are known to inhibit protein synthesis, induce the rapid phosphorylation of RAX followed by RAX-PKR association and activation of PKR. Therefore, cellular RAX may be a stress-activated, physiologic activator of PKR that couples transmembrane stress signals and protein synthesis.

Amino Acid Sequence↗
PubMed2000

Functional characterization of SR and SR-related genes in Caenorhabditis elegans.

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

Amino Acid Sequence↗
PubMed2006

Rotavirus proteins: structure and assembly.

Rotavirus is a major pathogen of infantile gastroenteritis. It is a large and complex virus with a multilayered capsid organization that integrates the determinants of host specificity, cell entry, and the enzymatic functions necessary for endogenous transcription of the genome that consists of 11 dsRNA segments. These segments encode six structural and six nonstructural proteins. In the last few years, there has been substantial progress in our understanding of both the structural and functional aspects of a variety of molecular processes involved in the replication of this virus. Studies leading to this progress using of a variety of structural and biochemical techniques including the recent application of RNA interference technology have uncovered several unique and intriguing features related to viral morphogenesis. This review focuses on our current understanding of the structural basis of the molecular processes that govern the replication of rotavirus.

Antigens, Viral↗
PubMed1999

Suppression of interferon response gene expression in cells persistently infected with mumps virus, and restoration from its suppression by treatment with ribavirin.

Persistent infections with mumps virus were established in human B-lymphoid cell line Akata and in the human chronic myelogenous leukaemia cell line K562. Even after IFN treatment a drastic decrease in STAT-1alpha (signal transducers and activators of transcription-1alpha), STAT-2 and p48 (ISGF-3gamma: IFN-stimulated gene factor-3gamma), which are closely correlated with the IFN-signaling pathway, was found in these persistently infected cells (Akata-MP1 and K-MTP). Therefore, the IFN-signaling pathway is thought to be defective in these persistently infected cells. In other words, most of the IFN-inducible genes in these cells persistently infected with mumps virus may not be able to respond to IFN treatment. Indeed, poor induction of 2',5'-oligoadenylate synthetase (2-5AS), dsRNA activated protein kinase (PKR), and MxA protein mRNAs were demonstrated in these cell lines after IFN treatment. Expression of MHC class-I antigen was also significantly reduced in the persistently infected cell lines as compared with that of uninfected control cells. HLA antigen was augmented by IFN-alpha in Akata and K562 cells, but not in persistently infected cells. Furthermore, suppression of IFN-induced 2-5AS induction and MHC class-I expression was restored by treatment of persistently infected cells with ribavirin through inhibition of virus replication. The result of restoration was also confirmed by IFN-induced STAT-1 induction in persistently infected cells treated with ribavirin.

Antiviral Agents↗
PubMed1994

Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.

Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.

Adenosine↗
PubMed2000

Intrahepatic mRNA expression of interferon-inducible antiviral genes in liver diseases: dsRNA-dependent protein kinase overexpression and RNase L inhibitor suppression in chronic hepatitis C.

As a part of the defense mechanism of the host to viral infection, interferons induce the transcription of several genes. These interferon-inducible genes contribute to the eradication of the viruses. Whereas some studies suggested the participation of a dsRNA-dependent protein kinase in the host reaction to hepatitis C virus infection, the involvement of other interferon-inducible genes has not been evaluated. Furthermore, there has been no analysis on the expression profile of multiple interferon-inducible genes. The aim of this study was to clarify the hepatic mRNA expression profile of interferon-inducible genes with a special concern to chronic hepatitis C. A total of 76 liver biopsy samples (28 with chronic hepatitis C, 10 with chronic hepatitis B, 9 with alcoholic liver disease, 14 with autoimmune hepatitis, 10 with primary biliary cirrhosis, and 5 of normal liver) were enrolled. The expression of the following genes was quantified by competitive reverse transcription-polymerase chain reaction and was compared according to the etiology; dsRNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (2,5-AS), latent cellular endoribonuclease (RNase L), RNase L inhibitor, and MxA. As a result, PKR mRNA was significantly overexpressed in the liver of chronic hepatitis C compared with those of other etiologies (P =.0178), and it correlated significantly with serum alanine transaminase values (r =.51, P =.0054). Also, the expression of the RNase L inhibitor showed a significant reduction in chronic hepatitis C (P =.0184). The expressions of 2,5-AS, RNase L, and MxA were not different significantly irrespective to the etiology. In conclusion, hepatic overexpression of PKR and reduced expression of RNase L inhibitor seem to contribute to the anti-HCV mechanism characteristically.

ATP-Binding Cassette Transporters↗
PubMed1999

Upregulation of STAT1 protein in cells lacking or expressing mutants of the double-stranded RNA-dependent protein kinase PKR.

The interferon (IFN)-inducible double-stranded (ds) RNA-dependent protein kinase PKR plays a role in the regulation of gene expression through its capacity to phosphorylate the translation initiation factor eIF-2 and to inhibit protein synthesis. In addition to translational control, PKR has been implicated in the regulation of gene expression at the transcriptional level. In this regard, we have reported that PKR participates in IFN-and dsRNA-mediated signaling pathways by interacting with and modulating the transcriptional activity of the signal transducer and activator of transcription STAT1 [Wong, A.H.-T., Tam, N.W.N., Yang, Y.-L., Cuddihy, A.R., Li, S., Kirchhoff, S., Hauser, H., Decker, T. & Koromilas, A.E. (1997) EMBO J. 16, 1291-1304]. Here we report that the STAT1 protein is upregulated in cells lacking PKR (PKR-/-) and in cells expressing dominant negative PKR mutants. This upregulation is specific for STAT1 as increased expression is not observed for other STAT proteins. The inhibitory effect of PKR on STAT1 expression is exerted at the post-translational level because PKR-/- cells exhibit higher STAT1 protein stability than PKR+/+ cells.

Animals↗
PubMed1999

The importance of internal loops within RNA substrates of ADAR1.

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, we investigated the role of internal loops within ADAR substrates. We observed that a dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (>/=six nucleotides) acted as helix ends, whereas smaller loops (</=four nucleotides) did not. Our work provides the first information about how secondary structure within ADAR substrates dictates selectivity, and suggests a rational approach for delineating minimal substrates for RNAs deaminated by ADARs in vivo.

Adenosine Deaminase↗
PubMed1993

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Animals↗
PubMed2001

A RHO GTPase-mediated pathway is required during P cell migration in Caenorhabditis elegans.

The Rho family of guanine triphosphate hydrolases controls various cellular processes, including cell migration. We describe here the demonstration of a role for a RhoA GTPase homologue during cell migration in Caenorhabditis elegans. We show that eliminating or reducing rho-1 gene function by using a dominant-negative transgene or dsRNA interference results in a severe defect in migration of hypodermal P cells to a ventral position. Biochemical and genetic data also suggest that unc-73, which encodes a Trio-like guanine nucleotide exchange factor, may act as an activator of rho-1 in the migration process. Mutations in let-502 ROCK, a homologue of a RhoA effector in mammals, also cause defects in P cell migration, suggesting that it may be one of several effectors acting downstream of rho-1 during P cell migration. Finally, we provide evidence to support the idea that other small Rac subfamily small GTPases act redundantly and in parallel to RHO-1 in this specific cell migration event.

Animals↗