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PubMed2006

GCSF receptor regulates antigen uptake and expression of cytokines and costimulatory molecules in dendritic cells.

RNA interference (RNAi), a process that specifically silences target gene expression, is a powerful technique to modulate cellular functions. In this study, we identified two small interference RNA (siRNA) sequences that can specifically and efficiently silence the expression of the granulocyte colony-stimulating factor receptor (GCSF-R) gene and achieved stable knockdown of GCSF-R using pFIV lentivirus containing the GCSF-R siRNA. GCSF-R knockdown significantly reduces the expression of IL-lalpha, IL-lbeta, IL-6, IL-10, H-2Kb, I-Ab, CD80 and CD86, and increases PDL1 and PDL2 expression, while IL-12p35, TGFbeta, TNFalpha and CD40 expression is unaltered. Furthermore, GCSF-R knockdown significantly changes the endocytosis and micro-pinocytosis abilities as well as surface expression of antigens of DC2.4 cells.

Animals↗

PubMed2004

The involvement of two cdc2-related kinases (CRKs) in Trypanosoma brucei cell cycle regulation and the distinctive stage-specific phenotypes caused by CRK3 depletion.

Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA(i)) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G(1)/S transition, whereas CRK3 plays an important role in catalyzing cells across the G(2)/M junction. A knockdown of CRK1 caused accumulation of cells in the G(1) phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G(2)/M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G(1) phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stage-specific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.

Animals↗

PubMed2004

Down-regulation amyloid beta-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA.

AIM: To investigate the pathogenesis of Abeta42 yielding and new drug targets as well as the possibility of RNA interference (RNAi) technique for treatment of Alzheimer disease (AD). METHODS: Human AD presenilin 1 (PS1) cDNA sequence was obtained from NCBI website. The three sites of RNAi action and one missense control site were selected in PS1 cDNA through online design of Ambion company. To confirm specificity of these sites, we conducted a BLAST search of the IMAGE EST library. The corresponding double-stranded DNA was used to construct pSilencer 3.1-H1 plasmid, which could transcribe small interference RNA (siRNA). Then, the pSliencer 3.1-H1 plasmids were transfected into CHO/PS1/APP cells with SuperFect transfection reagent. The cells have been transfected with the mutant PS1 and APP gene of AD. All the CHO/PS1/APP cells with pSliencer 3.1-H1 plasmids were screened out using G418. Transcripts of PS1 gene in CHO/PS1/APP were measured by RT-PCR, the contents of PS1 peptide and Abeta42 production inside CHO/PS1/APP cells were examined through Western blot and the Abeta42 change of secretion by CHO/PS1/APP was determined with ELISA. RESULTS: The site 3 of PS1 mRNA was inhibited by RNAi after 2 d. The effect was more obvious with the time. The peptide corresponding to PS1 gene and Abeta42 production in CHO/PS1/APP cells were both reduced after siRNA interfere for 3 d. Abeta42 secretion by CHO/PS1/APP cells began to reduce on d 3, and reached the most significance on d 5. There was a time-dependent relationship between the transcript of PS1 gene and the production of Abeta42 with RNAi action. CONCLUSION: PS1 is essential for g-secretase activity. Inhibition of the PS1 can decrease the levels of Abeta42. Some sites of PS1 mRNA, for example, the site 3, may serve as a new drug target and RNAi probably can be used for treatment of AD.

Alzheimer Disease↗

PubMed2005

Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes.

Short RNA regulatory molecules, microRNAs, and short interfering RNAs participate in a range of developmental gene networks by base-pairing with their target sequences. Consistent with these findings, genes required for the biogenesis and function of short interfering RNAs and microRNAs, dicer (dcr-1 in Caenorhabditis elegans) and argonaute homologs, are essential for development in diverse organisms, including C. elegans. We demonstrate that genes required for the function of short RNAs synergize with the retinoblastoma tumor suppressor homolog lin-35 in negative regulation of the nuclear divisions in the intestine of C. elegans. The level of cyclin E (cye-1) expression is critical for nuclear divisions in the intestine and is elevated in double mutants in lin-35 and RNA interference pathway genes. We propose that RNA interference-related pathways cooperate with retinoblastoma in transcriptional repression of endogenous genes, an example being cyclin E.

Animals↗

PubMed2003

[Progress of miRNA and its functions in eukaryotes].

Two major kinds of non-coding RNAs play important roles in eukaryotes. One is microRNA (miRNA), the other is small interference RNA (siRNA). miRNA, 19-25 nt in length, functions in regulation of gene expression and development. Many kinds of miRNAs and some proteins assemble in a complex, miRNP, where miRNA gives its function, siRNA directs the target mRNA in RNA interference (RNAi). Some distinct differences are existed between miRNA and siRNA. The regulation mechanism of miRNA may be conserved in eukaryotes.

Animals↗

PubMed2006

Formation of GW bodies is a consequence of microRNA genesis.

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.

Cytoplasmic Structures↗

PubMed2004

Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B.

Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the beta-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.

Animals↗

PubMed2005

Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA.

Inhibition of the up-regulated telomerase activity in cancer cells has previously been shown to slow cell growth but only after prior telomere shortening. Previously, we have reported that, unexpectedly, a hairpin short interfering RNA specifically targeting human telomerase RNA rapidly inhibits the growth of human cancer cells independently of p53 or telomere length and without bulk telomere shortening (Li, S., Rosenberg, J. E., Donjacour, A. A., Botchkina, I. L., Hom, Y. K., Cunha, G. R., and Blackburn, E. H. (2004) Cancer Res. 64, 4833-4840). Here we have demonstrated that such telomerase RNA knockdown in cancer cells does not cause telomere uncapping but rather induces changes in the global gene expression profile indicative of a novel response pathway, which includes suppression of specific genes implicated in angiogenesis and metastasis, and is distinct from the expression profile changes induced by telomere-uncapping mutant template telomerase RNAs. These cellular responses to depleting telomerase in human cancer cells together suggest that cancer cells are "telomerase-addicted" and uncover functions of telomerase in tumor growth and progression in addition to telomere maintenance.

Base Sequence↗

PubMed2006

Urokinase signaling through its receptor protects against anoikis by increasing BCL-xL expression levels.

The acquired capabilities of resistance to apoptotic cell death and tissue invasion are considered to be obligate steps in tumor progression. The binding of the serine protease urokinase (uPA) to its receptor (uPAR) plays a central role in the molecular events coordinating tumor cell adhesion, migration, and invasion. Here we investigate whether uPAR signaling may also prevent apoptosis following loss of anchorage (anoikis) or DNA damage. If nontransformed human retinal pigment epithelial cells are pre-exposed to uPA or to its noncatalytic amino-terminal region (residues 1-135), they exhibit a markedly reduced susceptibility to anoikis as well as to UV-induced apoptosis. This anti-apoptotic effect is retained by a uPA-derived synthetic peptide corresponding to the receptor binding domain and is inhibited by anti-uPAR polyclonal antibodies. Furthermore, the stable reduction of uPA or uPAR expression by RNA interference leads to an increased susceptibility to UV-, cisplatin-, and detachment-induced apoptosis. In particular, the level of uPAR expression positively correlates with cell resistance to anoikis. The protective ability of uPA is prevented by UO126, LY294002, by an MAPK targeting small interference RNA, and by a dominant negative Akt variant. Accordingly, incubation of retinal pigment epithelial cells with uPA elicits a time-dependent enhancement of MAPK and phosphatidylinositol 3-kinase activities as well as the transcriptional activation of Bcl-xL anti-apoptotic factor. Vice versa, the silencing of Bcl-xL expression prevents uPA protection from anoikis. In conclusion, the data show that ligand engagement of uPAR promotes cell survival by activating Bcl-xL transcription through the MEK/ERK- and phosphatidylinositol 3-kinase/Akt-dependent pathways.

Anoikis↗

PubMed2006

Insulin-stimulated Interaction between insulin receptor substrate 1 and p85alpha and activation of protein kinase B/Akt require Rab5.

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.

Animals↗

PubMed2005

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress.

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.

Adaptation, Physiological↗

PubMed2003

Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences.

RNA interference (RNAi) is a double-stranded (ds)RNA-inducible, sequence-specific RNA-degradation mechanism that operates as a natural antiviral system in plants and animals. Successful virus infection requires evasion or suppression of RNAi. Indeed, RNAi suppressor proteins have been identified in plant and animal viruses, although the molecular mechanism of silencing inhibition is still poorly understood. Because many RNA viruses encode dsRNA-binding proteins (dsRBPs) and as RNAi is triggered by the accumulation of dsRNAs, dsRBPs were examined to see if they inhibit RNAi. Here, it is shown that heterologous dsRBPs suppressed RNAi in plants, indicating that in natural host-virus interactions, pathogen-encoded dsRBPs could inactivate RNAi-mediated host defences.

Plant Proteins↗

PubMed2005

Small but mighty RNA-mediated interference in plants.

RNA silencing is a conserved phenomenon of regulation of gene expression by small RNAs derived from cleavage of double-stranded RNA (dsRNA). The present review deals with three overlapping modes of small RNA-mediated silencing particularly in plants. In case of post-transcriptional gene silencing (PTGS), Dicer, an endonuclease, cleaves dsRNA to produce approximately 21nt-long small interfering RNAs (siRNAs), which guide RISC, another nuclease complex, to destroy specific target mRNAs based on sequence complementarity with the siRNA. Another class of siRNAs of 25nt-long is also produced from dsRNA by Dicer, different from that generates 21nt-long siRNA. These longer siRNAs are probably involved in systemic silencing during PTGS and guide methylation of both DNA and histone, and induce heterochromatinization and consequent transcriptional repression of the targeted gene. Both siRNA-mediated PTGS and epigenetic modification of the genome are considered as defense mechanisms to protect against invading viruses, transposons or aberrantly expressing transgenes. Regulation of expression of endogenous genes is mediated by another class of 21nt-long small RNAs called microRNAs (miRNA). Genes encoding the miRNAs are present either in the intergenic regions, introns or coding regions of the plant genome. Cleavage of a stem-loop precursor transcript called pre-miRNA, by another class of Dicer generates miRNAs, which in association with nuclease complex similar to RISC, if not identical, either degrade target mRNA or cause translational repression. The applications of RNA silencing in functional genomics and crop improvement are discussed.

Gene Expression Regulation, Plant↗

PubMed2004

RNA silencing in viral infections: insights from poliovirus.

RNA interference or RNA silencing is a dsRNA guided mechanism that mediates sequence specific degradation of RNA. The recent demonstration that RNA interference can be used to inhibit virus replication has initiated an exciting field of research: first, as a potential novel antiviral therapeutic approach and, second, as a tool for dissecting virus-host interactions. Here we review and discuss the current data and perspectives on the use of RNA interference in the study of poliovirus as a model for positive strand RNA viruses.

Animals↗

PubMed2005

RNA interference-based strategies for metabolic syndrome treatment.

RNA interference is a naturally occurring cellular mechanism to inhibit the expression of specific gene products. The technical application of RNA interference offers great potential for the specific treatment of a huge variety of diseases including the metabolic syndrome, one of the most challenging threats to human health associated with our civilization. In order to develop novel and powerful strategies for the treatment of the metabolic syndrome, it is essential to define a set of specific gene products that may be targeted by RNA interference. Based on currently available in vitro and in vivo data, we discuss the feasibility of candidate genes involved in the pathophysiology of the metabolic syndrome as potential targets for a rational RNA interference based therapy in this review.

Animals↗

PubMed1996

The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb.

The protooncogene for Spi-1/PU.1 is an Ets-related transcription factor overexpressed during Friend erythroleukemia. The molecular basis by which Spi-1/PU.1 is involved in the erythroleukemic process remains to be elucidated. By using an immobilized protein binding assay, we have identified a 55-kDa protein as a putative partner of Spi-1/PU.1 protein. Microsequence analysis revealed that this 55-kDa protein was p54nrb (nuclear RNA-binding protein, 54 kDa) a RNA-binding protein highly similar to the splicing factor PSF (polypyrimidine tract-binding protein-associated splicing factor). In this paper, we show that Spi-1/PU.1 impedes the binding of p54nrb to RNA and alters the splicing process in vitro. Moreover, we present evidence that the transcriptional factor Spi-1/PU.1, unlike other Ets proteins, is able to bind RNA. Altogether, these results raise the intriguing possibility that the functional interference observed between Spi-1/PU.1 and RNA-binding proteins might represent a novel mechanism in malignant erythropoiesis.

Alternative Splicing↗

PubMed2004

RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing.

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.

Animals↗

PubMed2006

[Mitotic arrest of gastric cancer cells induced by silencing of STK15 gene].

OBJECTIVE: To investigate the role of STK15 in regulating mitosis of gastric cancer cells (MKN45) by gene silencing through RNA interference mechanism. METHODS: RNA interference technique was used to inhibit STK15 expression in MKN45 cells. The expression levels of STK15 mRNA and protein were measured by real-time quantitative RT-PCR and Western blot respectively and cell morphological changes were investigated by reverse microscopy. In addition, cell cycle distribution and cellular proliferation were determined by flow-cytometry and MTT assay respectively. Finally, the mitotic phenotype of MKN45 cells was studied by immunofluorescence staining and confocal microscopy. RESULTS: Silencing of STK15 gene by RNA interference was confirmed by marked decrease of STK15 mRNA and protein levels in the treated MKN45 cells. This silencing correlated with rounding of the cells, decreasing of DNA content in G(2) phase (P < 0.05) and a lowered proliferation index (P < 0.05), along with alterations of mitotic phenotype of MKN45 (P < 0.05). CONCLUSION: STK15 gene may play a key role in regulating cellular mitosis and its inhibition by RNA interference leading to mitosis arrest in MKN45 cells.

Adenocarcinoma↗