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PubMed2002

Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2005

SiRNA strategy against overexpression of extracellular matrix in diabetic retinopathy.

Increased synthesis of extracellular matrix (ECM) contributes to the development of vascular BM thickening, a prominent abnormality in diabetic retinopathy. RNA interference (RNAi) approach was used in this study to examine the effect of small interfering RNAs (siRNAs) for their ability to inhibit ECM-specific gene overexpression under high glucose condition in rat microvascular endothelial cells (RMECs). Four fibronectin (FN)-siRNAs, three collagen IV (Coll IV)-siRNAs, and four laminin (LM)-siRNAs, a total of 11 siRNAs were screened. RMECs were transfected with 10, 30, or 100 nm of each siRNAs in the presence of 8 microm lipofectin and subjected to analysis 72 hr after transfection. In long-term studies siRNA-transfected cells were examined after 12 days. Two FN siRNAs, two Coll IV siRNAs, and two LM siRNAs significantly reduced the respective target expressions. Findings from this study indicate that high glucose-induced abnormal expression of BM components may contribute to increased vascular permeability. SiRNA may be a useful tool in preventing excess vascular permeability, a characteristic feature of early diabetic retinopathy.

Animals↗
PubMed2004

Accelerated off-target search algorithm for siRNA.

MOTIVATION: Designing highly effective short interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi) is one of the hottest topics in molecular biology. The relationship between siRNA sequences and RNAi activity has been studied extensively to establish rules for selecting highly effective sequences. However, there is a pressing need to compute siRNA sequences that minimize off-target silencing effects efficiently and to match any non-targeted sequences with mismatches. RESULTS: The enumeration of potential cross-hybridization candidates is non-trivial, because siRNA sequences are short, ca. 19 nt in length, and at least three mismatches with non-targets are required. With at least three mismatches, there are typically four or five contiguous matches, so that a BLAST search frequently overlooks off-target candidates. By contrast, existing accurate approaches are expensive to execute; thus we need to develop an accurate, efficient algorithm that uses seed hashing, the pigeonhole principle, and combinatorics to identify mismatch patterns. Tests show that our method can list potential cross-hybridization candidates for any siRNA sequence of selected human gene rapidly, outperforming traditional methods by orders of magnitude in terms of computational performance. AVAILABILITY: http://design.RNAi.jp CONTACT: yamada@cb.k.u-tokyo.ac.jp.

Algorithms↗
PubMed2006

Improved transgene expression fine-tuning in mammalian cells using a novel transcription-translation network.

Following the discovery of RNA interference (RNAi) and related phenomena, novel regulatory processes, attributable to small non-protein-coding RNAs, continue to emerge. Capitalizing on the ability of artificial short interfering RNAs (siRNAs) to trigger degradation of specific target transcripts, and thereby silence desired gene expression, we designed and characterized a generic transcription-translation network in which it is possible to fine-tune heterologous protein production by coordinated transcription and translation interventions using macrolide and tetracycline antibiotics. Integration of siRNA-specific target sequences (TAGs) into the 5' or 3' untranslated regions (5'UTR, 3'UTR) of a desired constitutive transcription unit rendered transgene-encoded protein (erythropoietin, EPO; human placental alkaline phosphatase, SEAP; human vascular endothelial growth factor 121, VEGF(121)) production in mammalian cells responsive to siRNA levels that can be fine-tuned by macrolide-adjustable RNA polymerase II- or III-dependent promoters. Coupling of such macrolide-responsive siRNA-triggered translation control with tetracycline-responsive transcription of tagged transgene mRNAs created an antibiotic-adjustable two-input transcription-translation network characterized by elimination of detectable leaky expression with no reduction in maximum protein production levels. This transcription-translation network revealed transgene mRNA depletion to be dependent on siRNA and mRNA levels and that translation control was able to eliminate basal expression inherent to current transcription control modalities. Coupled transcription-translation circuitries have the potential to lead the way towards composite artificial regulatory networks, to enable complex therapeutic interventions in future biopharmaceutical manufacturing, gene therapy and tissue engineering initiatives.

Alkaline Phosphatase↗
PubMed2003

Activation of m-calpain is required for chromosome alignment on the metaphase plate during mitosis.

Calpains form a superfamily of Ca(2+)-dependent intracellular cysteine proteases with various isoforms. Two isoforms, micro- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca(2+) concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not mu-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.

Calcium↗
PubMed2006

[Inhibitory effect of blocking expression of human augmenter of liver regeneration (hALR) on proliferation of hepatocellular carcinoma cell line HepG2].

BACKGROUND & OBJECTIVE: Human augmenter of liver regeneration (hALR) is highly expressed in hepatocellular carcinoma (HCC), but rarely expressed in normal liver cells. ALR could stimulate the proliferation of HCC cell line in vitro, but has no effect on normal hepatocytes. This study was to investigate the inhibitory effect of blocking the expression of hALR on the proliferation of HCC cell line HepG2 using small interfering RNA (siRNA) targeting ALR and anti-ALR monoclonal antibody (McAb). METHODS: RNA interference (RNAi) plasmid pSIALR-A targeting hALR cDNA and control plasmid pSIALR-B were constructed and transfected into HepG2 cells, respectively. After transfection, the expression of green fluorescent protein was observed under a fluorescent microscope to calculate transfection efficiency, the protein level of hALR was measured by immunocytochemistry, meanwhile, the expression of hALR mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). (3)H-TdR incorporation approach was performed to detect the proliferation of HepG2 cells after transfection and after neutralization with anti-hALR McAb. RESULTS: hALR was expressed in HepG2 cells. Plasmids pSIALR-A and pSIALR-B were successfully constructed. Both immunocytochemistry and RT-PCR showed that pSIALR-A inhibited the expression of hALR in HepG2 cells significantly by 83% as compared with pSIALR-B. pSIALR-A specifically inhibited the growth of HepG2 cells after transfection. (3)H-TdR incorporation of pSIALR-A/HepG2 cells was significantly lower than that of pSIALR-B/HepG2 cells (67 687+/-6 548 vs. 104 807+/-5 713, P<0.05). Anti-hALR McAb markedly inhibited the autonomous growth of HepG2 cells (P<0.05). CONCLUSIONS: hALR is highly expressed in HepG2 cells. The short interfering RNA targeting hALR gene could specifically suppress the expression of hALR, and subsequently inhibit the growth of HepG2 cells. Anti-hALR McAb could partially inhibit the autonomous growth of HepG2 cells.

Antibodies, Monoclonal↗
PubMed2005

Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

Animals↗
PubMed2002

Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression.

Heterochronic genes control the timing of developmental programs. In C. elegans, two key genes in the heterochronic pathway, lin-4 and let-7, encode small temporally expressed RNAs (stRNAs) that are not translated into protein. These stRNAs exert negative post-transcriptional regulation by binding to complementary sequences in the 3' untranslated regions of their target genes. stRNAs are transcribed as longer precursor RNAs that are processed by the RNase Dicer/DCR-1 and members of the RDE-1/AGO1 family of proteins, which are better known for their roles in RNA interference (RNAi). However, stRNA function appears unrelated to RNAi. Both sequence and temporal regulation of let-7 stRNA is conserved in other animal species suggesting that this is an evolutionarily ancient gene. Indeed, C. elegans, Drosophila and humans encode at least 86 other RNAs with similar structural features to lin-4 and let-7. We postulate that other small non-coding RNAs may function as stRNAs to control temporal identity during development in C. elegans and other organisms.

3' Untranslated Regions↗
PubMed2005

A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice.

Auxin transport affects a variety of important growth and developmental processes in plants, including the regulation of shoot and root branching. The asymmetrical localization of auxin influx and efflux carriers within the plasma membrane establishes the auxin gradient and facilitates its transport. REH1, a rice EIR1 (Arabidopsis ethylene insensitive root 1)-like gene, is a putative auxin efflux carrier. Phylogenetic analysis of 32 members of the PIN family, taken from across different species, showed that in terms of evolutionary relationship, OsPIN1 is closer to the PIN1 family than to the PIN2 family. It is, therefore, renamed as OsPIN1 in this study. OsPIN1 was expressed in the vascular tissues and root primordial in a manner similar to AtPIN1. Adventitious root emergence and development were significantly inhibited in the OsPIN1 RNA interference (RNAi) transgenic plants, which was similar to the phenotype of NPA (N-1-naphthylphalamic acid, an auxin-transport inhibitor)-treated wild-type plants. alpha-naphthylacetic acid (alpha-NAA) treatment was able to rescue the mutated phenotypes occurring in the RNAi plants. Overexpression or suppression of the OsPIN1 expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio. Taken together, these data suggest that OsPIN1 plays an important role in auxin-dependent adventitious root emergence and tillering.

Base Sequence↗
PubMed2005

esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.

Chromatography, Gel↗
PubMed2004

siRNA relieves chronic neuropathic pain.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

Animals↗
PubMed2005

Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells.

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.

Animals↗
PubMed2002

RNA interference: advances and questions.

In animals and protozoa gene-specific double-stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post-transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasite Trypanosoma brucei represents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi in T. brucei and outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.

Animals↗
PubMed2003

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Animals↗
PubMed2003

Control of siRNA expression utilizing Cre-loxP recombination system.

Vector-mediated systems for specific siRNA expression in mammalian cells using pol III promoters allowing high level of transcription activity have been developed in the past years, widening the usage of RNA interference (RNAi). In this study, we controlled the pol III promoter (U6 promoter)-driven expression of siRNA using the Cre-loxP system. Our "Cre-On" siRNA-expression vector against firefly luciferase activity could be switched on only in the presence of Cre recombinase, which, in this study, was delivered directly from the medium into the cells as TAT-NLS-Cre, a fusion protein with TAT peptide (an Arg rich peptide derived from HIV) and nuclear localizing signal (NLS). Upon the addition of TAT-NLS-Cre, complete and functional siRNAs were generated and reporter activity was suppressed.

Integrases↗
PubMed2006

Causative role of oxidative stress in a Drosophila model of Friedreich ataxia.

Friedreich ataxia (FA), the most common form of hereditary ataxia, is caused by a deficit in the mitochondrial protein frataxin. While several hypotheses have been suggested, frataxin function is not well understood. Oxidative stress has been suggested to play a role in the pathophysiology of FA, but this view has been recently questioned, and its link to frataxin is unclear. Here, we report the use of RNA interference (RNAi) to suppress the Drosophila frataxin gene (fh) expression. This model system parallels the situation in FA patients, namely a moderate systemic reduction of frataxin levels compatible with normal embryonic development. Under these conditions, fh-RNAi flies showed a shortened life span, reduced climbing abilities, and enhanced sensitivity to oxidative stress. Under hyperoxia, fh-RNAi flies also showed a dramatic reduction of aconitase activity that seriously impairs the mitochondrial respiration while the activities of succinate dehydrogenase, respiratory complex I and II, and indirectly complex III and IV are normal. Remarkably, frataxin overexpression also induced the oxidative-mediated inactivation of mitochondrial aconitase. This work demonstrates, for the first time, the essential function of frataxin in protecting aconitase from oxidative stress-dependent inactivation in a multicellular organism. Moreover our data support an important role of oxidative stress in the progression of FA and suggest a tissue-dependent sensitivity to frataxin imbalance. We propose that in FA, the oxidative mediated inactivation of aconitase, which occurs normally during the aging process, is enhanced due to the lack of frataxin.

Aconitate Hydratase↗
PubMed2005

MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis.

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.

Contractile Proteins↗
PubMed2003

Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function.

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.

Arabidopsis↗