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PubMed2003

Antisense technologies. Improvement through novel chemical modifications.

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti-mRNA strategies can be distinguished. Firstly, the use of single stranded antisense-oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side-effects. Some of these problems have been solved in 'second generation' nucleotides with alkyl modifications at the 2' position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA-cleaving ribozymes and deoxyribozymes, and the use of 21-mer double-stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.

Animals↗

PubMed2005

Lymphoproliferative disorders: prospects for gene therapy.

The lymphoproliferative disorders represent a large group of diseases with a significant variation in presentation and clinical course. There has been a trend of increasing incidence for some of these disorders, and despite advances in therapies, a significant number of patients either respond poorly or have early relapses. For this reason there is a need to investigate novel therapies to be used either alone or as adjunct treatment in combination with conventional therapies. Gene therapy is a relatively new field that takes advantage of our increased understanding of molecular biology with the aim of treating a variety of diseases including cancer. It is defined as the introduction of genetic material into cells for therapeutic intent. Methods to improve gene delivery efficiency have been the focus of a large amount of research and to date the optimal procedure uses viruses such as oncoretroviruses, lentiviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. There are four main gene therapy strategies that might be used for the treatment of lymphoproliferative disorders. First, immunotherapy using tumour vaccines or techniques to enhance the function of immune effector cells has been investigated with some success in patients with B-cell malignancies. Second, the introduction of prodrug-activated 'suicide' genes into cells has been explored, in particular in patients with post-transplantation lymphoproliferative disease. Third, direct lysis of tumour cells using viruses shows some early promise, especially in the treatment of B-cell disorders by manipulating the measles virus to target the CD20 antigen. Finally, anti-gene strategies such as anti-sense therapy, ribozymes, and most recently RNA interference, could be used to suppress expression of specific target genes. RNA interference in particular has tremendous potential and has been studied in the context of anaplastic large cell lymphoma as well as Epstein-Barr virus-associated malignancies. Whilst we are still in the early days of this field and to date results have been modest, there is still a significant potential for gene therapy to play a role in the future treatment of these disorders.

Animals↗

PubMed2006

Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells.

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.

Animals↗

PubMed2004

miRNAs on the move: miRNA biogenesis and the RNAi machinery.

Recent advances have led to a more detailed understanding of RNA interference and its role in microRNA biogenesis and function. Primary microRNA transcripts are processed by the RNaseIII nuclease, Drosha, and are exported from the nucleus by Exportin-5. Dicer cleaves microRNAs into their mature forms, which can be incorporated into effector complexes that mediate gene silencing activities. The 3' two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway. MicroRNA functional analyses and genetic and biochemical interrogation of components of the pathway are starting to provide a glimpse at the range of biological processes and phenomena regulated by RNA interference.

Animals↗

PubMed2007

Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax.

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.

Animals↗

PubMed2006

Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis.

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-beta-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

Adenosine Triphosphate↗

PubMed2006

RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

Amino Acid Motifs↗

PubMed2005

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome.

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.

Acyltransferases↗

PubMed2005

Small inhibitory RNA--a tool for credentialing candidate genes.

The effect of a drug is related to the interaction of the numerous gene products that participate in a drug pathway. The clinical importance of genetic variability on the overall outcome of most drug pathways remains to be determined. As a result, there is a need to efficiently identify the genes that not only participate in the pathway but also play a significant role in regulating the effects of a drug. The emerging technique of specific gene silencing through RNA interference initiated by small inhibitory RNA may prove to be a useful and powerful tool in the credentialing of candidate genes in drug pathways.

Animals↗

PubMed2006

Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei.

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).

Animals↗

PubMed2006

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

Animals↗

PubMed2003

Deficiency of Caenorhabditis elegans RecQ5 homologue reduces life span and increases sensitivity to ionizing radiation.

Gene expression and RNA interference phenotypes were investigated for a Caenorhabditis elegans homologue (Ce-RCQ-5) of human RecQ5 protein. Expression of the mRNA was observed by in situ hybridization from earliest embryogenesis and gradually decreased during late embryogenesis. Ce-RCQ-5 was immuno-localized in the nuclei of embryos, germ cells, and oocytes and also in the nuclei of various somatic cells of larvae and adults. Despite ubiquitous expression in postembryonic cells, RCQ-5 protein expression was highest in intestinal cells, which was confirmed by tagging the gene expression with green fluorescence protein. When endogenous Ce-rcq-5 gene expression was inhibited by RNA interference, no clear phenotypes were observed during development. However, C. elegans life span was reduced by 37% due to RNA interference of rcq-5 gene, suggesting its possible role in maintenance of genomic stability, as has been ascribed to other RecQ family DNA helicases. In addition, C. elegans became significantly more sensitive to ionizing radiation after inhibition of rcq-5 gene expression, indicating an involvement of C. elegans RCQ-5 in a cellular response to DNA damage, possibly in DNA repair.

Animals↗

PubMed2004

Mechanism of Dronc activation in Drosophila cells.

Proteolytic processing is required for the activation of most caspases. However, recent reports have suggested that the activation of the mammalian initiator caspase caspase-9 occurs during dimerization rather than after processing. Previously, we reported that, in normal living Drosophila S2 cells, the initiator caspase Dronc is continuously processed to a 40 kDa form we called Pr1 and that, during apoptosis, a second processed form of 37 kDa is also observed, which we called Pr2. In this study, we determined that Dronc Pr1 is the result of Dronc autoprocessing at amino acid E352, whereas Pr2 results from Drice cleaving full-length Dronc at amino acid D135. By using purified recombinant proteins and expressing Dronc cleavage mutants in S2 cells, we determined that autoprocessing at E352 is crucial for Dronc caspase activity, whereas Drice cleavage at D135 has little effect on Dronc activity. Suppression of the oligomerizing factor Dark by RNA interference revealed that Dark is required for Dronc autoprocessing at E352, whereas RNA interference of the effector caspase Drice revealed that Drice is also required for apoptosis in S2 cells. These results provide the first details of the mechanisms regulating initiator caspase activation in an invertebrate organism.

Animals↗

PubMed2004

Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis.

Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.

Amino Acid Sequence↗

PubMed2005

Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

Animals↗

PubMed2002

RNAi in human cells: basic structural and functional features of small interfering RNA.

We investigated the mechanism of RNA interference (RNAi) in human cells. Here we demonstrate that the status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells were phosphorylated, and 3' end biotin groups were not efficiently removed. We found no requirement for a perfect A-form helix in siRNA for interference effects, but an A-form structure was required for antisense-target RNA duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen did not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Animals↗

PubMed2003

Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells.

Gene silencing mediated by double-stranded RNA is a sequence-specific RNA degradation mechanism highly conserved in eukaryotes that serves as an antiviral defense pathway in both plants and Drosophila. Short interfering RNAs (siRNAs), the 21- to 23-nt double-stranded intermediates of this natural defense mechanism, are becoming powerful tools for reducing gene expression and countering viral infection in a variety of mammalian cells. Here we report the use of siRNAs to target reporter gene expression and viral DNA accumulation in cultured plant cells. Transient expression of reporter genes encoding either GFP or red fluorescent protein from Discosoma was specifically reduced by 58% and 47%, respectively, at 24 h after codelivery of cognate siRNAs in BY2 protoplasts. In contrast to mammalian systems, the siRNA-induced silencing of GFP expression was transitive as indicated by the presence of siRNAs representing parts of the target RNA outside the region homologous to the triggering siRNA. Codelivery of an siRNA designed to target the mRNA encoding the replication-associated protein (AC1) of the geminivirus African cassava mosaic virus (ACMV) from Cameroon blocked AC1 mRNA accumulation by approximately 91% and inhibited accumulation of the ACMV genomic DNA by approximately 66% at 36 and 48 h after transfection. As with siRNA-induced reporter gene silencing, the siRNA targeting ACMV AC1 was specific and did not affect the replication of East African cassava mosaic Cameroon virus. This report demonstrates the occurrence of siRNA-mediated suppression of gene expression in cultured plant cells and that siRNA can interfere with and suppress accumulation of a nuclear-replicated DNA virus.

Base Sequence↗