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At least 1,081 records · Page 60Linked to original sources
PubMed2002

MRG-1, a mortality factor-related chromodomain protein, is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans.

We identified MRG-1, a Caenorhabditis elegans chromodomain-containing protein that is similar to the human mortality factor-related gene 15 product (MRG15). RNA-mediated interference (RNAi) of mrg-1 resulted in complete absence of the germline in both hermaphrodite and male adults. Examination of the expression of PGL-1, a component of P granules, revealed that two primordial germ cells (PGCs) are produced during embryogenesis in mrg-1(RNAi) animals, but these PGCs cannot undergo mitotic proliferation, and they ultimately degenerate during post-embryonic development. Zygotic RNAi experiments using RNAi-deficient hermaphrodites and wild-type males demonstrated that MRG-1 functions maternally. Moreover, immunoblot analysis using mutant animals with germline deficiencies indicated that MRG-1 is synthesized predominantly in oocytes. These results suggest that MRG-1 is required maternally to form normal PGCs with the potential to start mitotic proliferation during post-embryonic development.

Amino Acid Sequence↗
PubMed2000

Depletion of a Cks homolog in C. elegans embryos uncovers a post-metaphase role in both meiosis and mitosis.

In eukaryotic cells, the key regulators of cell-cycle transitions are the cyclin-dependent kinases (CDKs). The best studied CDK is a component of the M-phase promoting factor (MPF), which promotes entry into and progression through meiosis and mitosis. One of the enduring mysteries of the MPF complex has been the role of Cks/Suc1, a highly conserved member of the cell-cycle machinery in eukaryotes [1,2]. Cks has been proposed to be involved in activation of MPF [3], general interactions of MPF with its mitotic substrates [4] and/or inactivation of MPF [5,6]. We identified two Cks homologs in the genome of Caenorhabditis elegans and used RNA-mediated interference (RNAi) to investigate their roles in development. Whereas cks-2(RNAi) embryos display no apparent defects, cks-1(RNAi) embryos display defects in both meiosis and mitosis. Specifically, cks-1(RNAi) embryos fail to exit M phase properly. We propose that CKS-1 has an essential role in the inactivation of MPF during early C. elegans embryogenesis.

Animals↗
PubMed2003

In vitro analysis of RNA interference in Drosophila melanogaster.

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.

Animals↗
PubMed2005

Functional genomics using high-throughput RNA interference.

RNA interference (RNAi) describes the post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into cells. Application of RNAi in experimental systems has provided a great leap forward in the elucidation of gene function. To facilitate large-scale functional genomics studies using RNAi, several high throughput approaches have been developed based on microarray or microwell assays. Recent establishment of large libraries of RNAi reagents combined with a variety of detection assays further opens the door for genome-wide screens of gene function in mammalian cells.

Animals↗
PubMed2006

Harnessing in vivo siRNA delivery for drug discovery and therapeutic development.

The use of RNA interference (RNAi) is spreading rapidly to nearly every aspect of biomedical research. The gene silencing capability of RNAi is being used to study individual gene's biological function and role in biochemical pathways. However, the efficacy of RNAi depends upon efficient delivery of the intermediates of RNAi, short interfering RNA (siRNA) and short hairpin RNA (shRNA) oligonucleotides. The delivery challenge is even greater when the aim is to inhibit the expression of target genes in animal models. Although i n vivo delivery of siRNA is complicated and challenging, recent results are encouraging. In this review, the latest developments of in vivo delivery of siRNA and the crucial issues related to this effort are addressed.

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↗
PubMed2006

Genetic engineering of Nicotiana tabacum for reduced nornicotine content.

UNLABELLED: Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco. KEYWORDS: Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.

Cytochrome P-450 Enzyme System↗
PubMed2002

RNA interference: applicability in tick research.

The rapid development of new genetic tools has boosted the gene discovery machinery. RNA interference (RNAi), a gene silencing process, has been recently used in several eukaryotic organisms to elucidate the function(s) of unknown genes and biochemical pathways. We used the dsRNA technique in Amlyomma americanum female ticks to test the applicability of the RNAi approach in ticks. Incubation of tick salivary glands (TSGs) in vitro and in vivo injection into whole female ticks with histamine binding protein (HBP) dsRNA led to a reduction in the HBP transcripts in the dsRNA treated groups. The dsRNA-injected ticks had a profound difference in their feeding pattern compared to control ticks that might reflect an increase in local histamine concentrations at the feeding sites. To our knowledge, this is the first RNAi study in ticks. In conclusion, RNAi can be applied in ticks and might be used to test the function of key proteins crucial for avoiding host defense at the tick-host interface.

Animals↗
PubMed2002

Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission.

The sec6/8, or exocyst, complex is implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane. Genetic analyses have been done primarily in yeast, where mutation of the eight protein subunits similarly disrupts polarized vesicle fusion. The goal of this study was to assay the sec6/8 complex in Drosophila, and specifically to test its widely hypothesized functions in synaptogenesis and neurotransmission. We used a transgenic RNAi approach to remove the most highly conserved complex component, Drosophila sec10 (dSec10). Ubiquitous dSec10 RNAi resulted in early postembryonic lethality, demonstrating that dSec10 is essential. Surprisingly, tissue-specific dSec10 RNAi revealed no essential requirement in nervous system, musculature, gut or epidermis. Assays of polarized secretion in all these tissues failed to reveal any role for dSec10. In particular, the neuromuscular synapse showed no defects in morphogenesis or vesicle trafficking/fusion underlying neurotransmission. The essential requirement for dSec10 was restricted to the ring gland, the Drosophila organ specialized for endocrine function. The developmental arrest of dSec10 RNAi animals was partially rescued by feeding ecdysone, suggesting dSec10 mediates steroid hormone secretion. We conclude that dSec10 has no detectable role in most forms of polarized trafficking/exocytosis, including neurotransmission, but rather is essential for endocrine secretion.

Animals↗
PubMed2000

RNA interference: genetic wand and genetic watchdog.

In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi.

Animals↗
PubMed2000

Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.

Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

Animals↗
PubMed2001

Role for a bidentate ribonuclease in the initiation step of RNA interference.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Animals↗
PubMed2004

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1.

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.

Amino Acid Sequence↗
PubMed2005

RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription.

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.

Argonaute Proteins↗
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↗
PubMed2006

Prevalence of off-target effects in Drosophila RNA interference screens.

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

Animals↗
PubMed2007

Slicer and the argonautes.

Though they started out as somewhat mysterious components of the RNAi effector complexes, Argonaute proteins have since taken center stage in RNAi gene silencing. They interact with small RNAs to effect gene silencing in all RNAi-related pathways known so far. We will review the dramatic advances in our understanding of the role of the Argonautes in RNAi through studies of their structure and function.

Amino Acid Sequence↗
PubMed2006

RNA interference machinery influences the nuclear organization of a chromatin insulator.

RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. We present evidence for a functional relationship between RNAi and the gypsy insulator of D. melanogaster. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression.

Animals↗