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Results for “RNAi pathway”

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At least 163 records · Page 9Linked to original sources
PubMed2006

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Animals↗
PubMed2004

Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena.

Genome-wide DNA elimination accompanies development of the somatic macronucleus from the germ-line micronucleus during the sexual process of conjugation in the ciliated protozoan Tetrahymena thermophila. Small RNAs, referred to as "scan RNAs" (scnRNAs), that accumulate only during conjugation are highly enriched in the eliminated sequences, and mutations that prevent DNA elimination also affect the accumulation of scnRNAs, suggesting that an RNA interference (RNAi)-like mechanism is involved in DNA elimination. Histone H3 that is methylated at lysine 9 (K9) is a hallmark of heterochromatin and, in Tetrahymena, is found only in developing macronuclei (anlagen) in association with chromatin containing the sequences undergoing elimination. In this article, we demonstrate that a mutation in the TWI1 gene that eliminates the accumulation of scnRNAs also abolishes H3 methylation at K9. We created mutant strains of Tetrahymena in which the only major H3 contained a K9Q mutation. These mutants accumulated scnRNAs normally during conjugation but showed dramatically reduced efficiency of DNA elimination. These results provide strong genetic evidence linking an RNAi-like pathway, H3 K9 methylation, and DNA elimination in Tetrahymena.

Animals↗
PubMed2026

A horizontally transferred bacterial gene for pantothenic acid biosynthesis regulates diapause and reproduction in the spider mite Amphitetranychus viennensis.

Horizontal gene transfer (HGT) has contributed substantially to the evolution of arthropod genomes, yet the functional significance of many horizontally acquired genes remains poorly understood. The hawthorn spider mite, Amphitetranychus viennensis, is a devastating agricultural pest whose high fecundity and overwintering diapause afford its exceptional ecological resilience. Through a genome-wide screen, we identified 37 high-confidence horizontally transferred genes (HTGs) in A. viennensis. Among these candidates, we prioritized AvPBL, a gene encoding pantothenate-β-alanine ligase, for functional characterization because it controls the rate-limiting step of a distinctly non-metazoan pantothenic acid (vitamin B5) biosynthesis pathway. RNAi-mediated suppression of AvPBL significantly reduced transcript abundance and endogenous pantothenic acid levels, triggering a 23.7% reduction in cumulative fecundity and severely compromising the mites' ability to enter winter diapause. Importantly, exogenous pantothenic acid supplementation rescued these reproductive and diapause defects, directly linking the observed phenotypes to the disruption of pantothenic acid biosynthesis. Our results demonstrate that the horizontally transferred bacterial gene AvPBL has been functionally integrated into the endogenous metabolic network of A. viennensis, playing a critical role in vitamin B5 biosynthesis, reproduction, and diapause regulation. These findings provide direct evidence that horizontally acquired metabolic genes can shape key life-history traits and drive adaptive evolution in arthropods.

Amphitetranychus viennensis↗
PubMed2006

Intronic microRNA (miRNA).

Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Changes in these sequences often manifest themselves in clinical and circumstantial malfunction. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) with complete or partial complementarity, are useful for the design of new therapies against cancer polymorphisms and viral mutations. Currently, many varieties of miRNA are widely reported in plants, animals, and even microbes. Intron-derived microRNA (Id-miRNA) is a new class of miRNA derived from the processing of gene introns. The intronic miRNA requires type-II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of Id-miRNA have been identified in C elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in not only human and mouse cells, but in also zebrafish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of intron-mediated gene silencing via functional miRNA in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Journal Article↗
PubMed2006

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.

Amino Acid Sequence↗
PubMed2004

ADAR1 RNA deaminase limits short interfering RNA efficacy in mammalian cells.

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.

Adenosine Deaminase↗
PubMed2005

Perspective: machines for RNAi.

RNA silencing pathways convert the sequence information in long RNA, typically double-stranded RNA, into approximately 21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. Here, we review our current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression.

Animals↗
PubMed2006

Autoimmune targeting of key components of RNA interference.

RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.

Animals↗
PubMed2006

The RNA interference pathway: a new target for autoimmunity.

Many intracellular macromolecular complexes that are involved in the production or degradation of RNAs are targeted by autoantibodies in systemic autoimmune diseases. RNA interference (RNAi) is a recently characterized gene silencing pathway by which specific mRNAs are either degraded or translationally suppressed. In a recent issue of Arthritis Research and Therapy, Andrew Jakymiw and colleagues reported that the enigmatic Su autoantigen complex contains key components of the RNAi machinery. Anti-Su autoantibodies from both human patients with rheumatic diseases and a mouse model of autoimmunity recognize the endonucleolytic Argonaute and Dicer proteins, both crucial enzymes of the RNAi pathway. These data raise the question of how the anti-Su response is triggered. So far, it is unknown whether molecular modifications may be involved, as has been proposed for other intracellular autoantigens. The implication of RNAi in anti-viral defence may suggest a role for virus infection in this process.

Animals↗
PubMed2003

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines.

BACKGROUND: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. METHODS: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. RESULTS: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. CONCLUSIONS: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

Animals↗
PubMed2004

Induction and biochemical purification of RNA-induced silencing complex from Drosophila S2 cells.

The discovery of RNA interference (RNAi) has greatly simplified the process of suppressing genes in many experimental systems, including Caenorhabditis elegans, Drosophila, and mammalian cells. A sequence-specific nuclease complex, called the RNA-induced silencing complex (RISC), can be purified from cells undergoing RNAi. RISC shows RNase activity when exposed to RNAs homologous to the input double-stranded RNA (dsRNAs) but lacks activity in the presence of nontargeted RNAs. We describe the induction of RNAi by dsRNA in cultured Drosophila Schneider-2 (S2) cells and detail procedures for RISC purification from these cells. This purification approach has allowed us to identify several RISC components, including siRNAs, Argo naute 2 (Ago-2), Drosophila Fragile X related protein (dFXR), Vasa intronic gene (VIG), and the micrococcal nuclease family member Tudor-SN (Drosophila CG7008). RNAi is carried out by an endogenous pathway important for normal development in many organisms. In fact, organisms express hundreds of different microRNAs (miRNAs), small hairpin RNAs that function through the RNAi pathway to suppress expression of endogenous genes. The function of miRNAs is poorly understood, and most of their targets are unknown. Purified RISC complexes contain short interfering RNAs and endogenously expressed miRNAs and will be useful for studying many aspects of the RNAi machinery.

Animals↗
PubMed2005

Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

Increasing evidence indicates that RNA interference (RNAi) may be used to provide antiviral immunity in mammalian cells. Human micro (mi)RNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN)-induced protein kinase R (PKR) but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si) and micro (mi)RNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

HIV↗
PubMed2004

The MAP kinase pathway is required for entry into mitosis and cell survival.

In this communication, we examined the role of the MAP kinase pathway in the G2/M phase of the cell cycle. Activation of the Plk1 and MAP kinase pathways was initially evaluated in FT210 cells, which arrest at G2 phase at the restrictive temperature (39 degrees C), due to a mutation in the cdc2 gene. Previous studies had shown that these cells enter mitosis at the nonpermissive temperature upon incubation with okadaic acid, a protein phosphatase 1 and 2A inhibitor. We show that treatment of FT210 cells at 39 degrees C with okadaic acid activated Plk1, as shown by hyperphosphorylation and elevated protein kinase activity, and also induced activation of the MAP kinase pathway. The specific Mek inhibitor PD98059 antagonized the okadaic acid-induced activation of both Plk1 and MAP kinases. This suggests that activation of the MAP kinase pathway may contribute to the okadaic acid-induced activation of Plk1 in FT210 cells at 39 degrees C. We also found that PD98059 strongly attenuated progression of HeLa cells through mitosis, and active Mek colocalizes with Plk1 at mitotic structures. To study the potential function of the MAP kinase pathway during mitosis, RNAi was used to specifically deplete five members of this pathway (Raf1, Mek1/2, Erk1/2). Each of these five protein kinases is required for cell proliferation and survival, and depletion of any of these proteins eventually leads to apoptosis. Treatment with Mek inhibitors also inhibited cell proliferation and caused apoptosis. A dramatic increase of Plk1 activities and a moderate increase of Cdc2 activities in Raf1-depleted cells indicate that Raf1-depleted cells arrest in the late G2 or M phase. Mek1 and Erk1 depletion also caused cell cycle arrest at G2, suggesting that these enzymes are required for the G2/M transition, whereas the loss of Mek2 or Erk2 caused arrest at G1.

Base Sequence↗
PubMed2006

Analysis of cell-based RNAi screens.

RNA interference (RNAi) screening is a powerful technology for functional characterization of biological pathways. Interpretation of RNAi screens requires computational and statistical analysis techniques. We describe a method that integrates all steps to generate a scored phenotype list from raw data. It is implemented in an open-source Bioconductor/R package, cellHTS (http://www.dkfz.de/signaling/cellHTS). The method is useful for the analysis and documentation of individual RNAi screens. Moreover, it is a prerequisite for the integration of multiple experiments.

Quality Control↗
PubMed2006

A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm.

We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.

Amino Acid Sequence↗
PubMed2006

From genome to phenome--RNAi library screening and hit characterization using signaling pathway analysis.

Comprehensive, high-throughput analysis of gene function using RNA interference (RNAi)-based screens is emerging as a significant step forward for preliminary drug-target identification. Until quite recently drug target identification depended heavily on the analysis of changes in gene expression, which in turn needed to be correlated with gene function. The promise of obtaining preliminary 'gene to phenotype' information using a single high-throughput platform is propelling major investment in this area by biotechnology and pharmaceutical companies. This review discusses recent developments in the use of RNAi library screens, and the various assay formats and techniques currently in use for data analysis downstream of such screens.

Animals↗
PubMed2005

RNAi mechanisms in Caenorhabditis elegans.

RNA interference (RNAi) is a form of gene silencing induced by double stranded RNA (dsRNA) that is processed into short interfering RNAs (siRNAs). RNAi can induce both post-transcriptional and transcriptional gene silencing. In Caenorhabditis elegans, there are several distinct pathways where post-transcriptional or/and transcriptional RNAi mechanisms are involved. RNAi in C. elegans is also systemic and heritable. This review will discuss RNAi related pathways, features of RNAi in C. elegans and possibilities of endogenous gene regulation by RNAi.

Animals↗