Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNAi pathway”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed2005

RNAi and RNA-based regulation of immune system function.

Gene regulation by short RNAs is a ubiquitous and important mode of control. MicroRNAs are short, single-strand RNAs that bind with partial complementarity to the 3' untranslated region of several genes to silence their expression. This expanding class of endogenous short RNAs are evolutionarily conserved and participate in control of development and cell-specific gene function. Several of these microRNAs have been cloned uniquely from mammalian lymphocytes suggesting specialized roles in lymphocyte development and function. In addition, several genes linked to RNAi in lower eukaryotes have mammalian homologs with specialized roles in adaptive immunity. For example, in worms, the nonsense-mediated decay (NMD) and RNAi pathways appear to be intricately linked. NMD plays a key role in regulating antigen-receptor expression in lymphocytes and there are mammalian homologs for factors identified in worms that appear to be common in both RNAi and NMD pathways. On the other hand, RNA editing and RNAi have an inverse relationship and RNA editing has an important role in viral immunity. These observations indicate unique roles for dsRNAs in the mammalian immune system.

APOBEC-1 Deaminase↗
PubMed2002

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

Amino Acid Sequence↗
PubMed2004

Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing.

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells.

Animals↗
PubMed2004

Eukaryotic gene regulation by targeted chromatin re-modeling at dispersed, middle-repetitive sequence elements.

RNA interference might have evolved to minimize the deleterious impact of transposable elements and viruses on eukaryotic genomes, because mutations in genes within the RNAi pathway cause mobilization of transposons in nematodes and flies. Although the first examples of RNAi involved post-transcriptional gene silencing, recently the pathway has been shown to act at the transcriptional level. It does so by establishing a chromatin configuration on the target DNA that has many of the hallmarks of heterochromatin, thus preventing its transcription. Members of dispersed, repeated sequence families appear to have been utilized by the RNAi machinery to regulate nearby genes in yeast. The unusual genomic distribution of three repeated element families in the chicken, fruit-fly and nematode genomes prompts speculation that some of these repeats have been co-opted to control gene expression, either locally or over extended chromosomal domains.

Animals↗
PubMed2004

RNA interference and the use of small interfering RNA to study gene function in mammalian systems.

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

Animals↗
PubMed2006

Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi.

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

Animals↗
PubMed2002

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

Amino Acid Motifs↗
PubMed2003

ago1 and dcr1, two core components of the RNA interference pathway, functionally diverge from rdp1 in regulating cell cycle events in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+ and dcr1+ but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+ and dcr1+ are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+ is dispensable for this process, the functions of ago1+ and dcr1+ in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+ is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+ function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.

Adenosine Triphosphatases↗
PubMed2006

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells.

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.

Animals↗
PubMed2005

RNA interference and heterochromatin in the fission yeast Schizosaccharomyces pombe.

Fission yeast is a useful model for RNA interference because it has single-copy genes for components of the RNAi pathway such as argonaute, dicer and RNA-dependent RNA polymerase (RdRP). Functions for RNAi revealed in S. pombe, such as heterochromatic silencing and chromosome segregation, are likely to be ancient because they are shared with some other eukaryotes. The underlying mechanisms are being rapidly unraveled.

Cell Division↗
PubMed2005

RNAi-directed assembly of heterochromatin in fission yeast.

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.

Animals↗
PubMed2006

Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation.

DNA methylation has important functions in stable, transcriptional gene silencing, immobilization of transposable elements and genome organization. In Arabidopsis, DNA methylation can be induced by double-stranded RNA through the RNA interference (RNAi) pathway, a response known as RNA-directed DNA methylation. This requires a specialized set of RNAi components, including ARGONAUTE4 (AGO4). Here we show that AGO4 binds to small RNAs including small interfering RNAs (siRNAs) originating from transposable and repetitive elements, and cleaves target RNA transcripts. Single mutations in the Asp-Asp-His catalytic motif of AGO4 do not affect siRNA-binding activity but abolish its catalytic potential. siRNA accumulation and non-CpG DNA methylation at some loci require the catalytic activity of AGO4, whereas others are less dependent on this activity. Our results are consistent with a model in which AGO4 can function at target loci through two distinct and separable mechanisms. First, AGO4 can recruit components that signal DNA methylation in a manner independent of its catalytic activity. Second, AGO4 catalytic activity can be crucial for the generation of secondary siRNAs that reinforce its repressive effects.

Amino Acid Sequence↗
PubMed2004

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi.

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.

Animals↗
PubMed2003

Asymmetry in the assembly of the RNAi enzyme complex.

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Adenosine Diphosphate↗
PubMed2006

Testing the efficacy of RNA interference in Haemonchus contortus.

RNA interference (RNAi) is widely used in Caenorhabditis elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could also be used on the strongylid parasitic nematode Haemonchus contortus to study gene function. Eleven genes were targeted in L1 and exsheathed L3 H. contortus larvae with RNAi methodologies which have been shown to be effective in C. elegans and parasitic nematodes-feeding, soaking and electroporation. Reverse transcriptase-PCR and, where possible, protein assays were carried out to examine decreases in mRNA and protein levels. RNAi soaking in dsRNA to beta-tubulin and sec-23, a gene involved in vesicle transport, resulted in specific decreases in mRNA levels in exsheathed L3 larvae. No signs of specific decreases in expression levels were observed for the other nine genes tested. Following electroporation of dsRNA in L1 stage larvae, significant decreases were observed for two out of four genes tested. These findings suggest that the RNAi pathway is functional in H. contortus and that, under certain conditions, it is possible to suppress gene expression by RNAi. However, it only works on a limited number of genes and in some cases the effect is small and difficult to reproduce. This indicates that the RNAi approaches established for C. elegans and other nematodes have limited efficacy in H. contortus. This may reflect differences between nematode species in dsRNA uptake and transport into cells and between cells.

Animals↗
PubMed2006

Lessons from Nature: microRNA-based shRNA libraries.

Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.

Animals↗
PubMed2004

Those interfering little RNAs! Silencing and eliminating chromatin.

RNA interference (RNAi) is widely used for knocking down expression of genes of interest and in systematic screens for desired phenotypes. In post-transcriptional gene silencing, double-stranded RNA triggers are processed to small interfering RNAs, which act to seek out and destroy homologous transcripts. A variety of organisms utilise the RNAi pathway to silence expression of potentially harmful endogenous mobile elements and to eliminate unnecessary sequences. In plants and fission yeast, RNAi can also mediate chromatin-based silencing resulting in transcriptional shutdown of homologous transcription units (transcriptional gene silencing) and the formation of centromeric heterochromatin. In metazoans, the expression of non-coding RNAs is often associated with the formation of silent chromatin domains but it remains to be determined if RNAi is involved.

Animals↗
PubMed2006

[Components and assembly of RNA-induced silencing complex].

Degradation of homologous RNA in RNA interference is carried out by functional RNA-induced silencing complex (RISC). RISC contains Dicer, Argonaute proein, siRNA and other components. Researching structures and functions of these components is primary important for understanding assembly and functional mechanism of RISC, as well as the whole RNAi pathway. Recent research works showed that Dicer, containing RNaseIII domain, is responsible for production of siRNA at the beginning of RNAi, and guarantees the stability of RISC intermediate in assembly process. As the core component of RISC, Argonaute protein functions as slicer to cleave target RNA and offers the binding site of siRNA in RISC assembly, which are depended on PIWI domain and PAZ domain separately. Although there is only one strand of siRNA that is the guider of RISC, the double stranded structural character of siRNA is determinant of RNAi. Except those, there are still other components with unknown functions in RISC. The knowledge about RISC components and assembly now, is basis of a presumed RISC assembly model.

Animals↗