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PubMed2004

Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane.

EHD1 has been implicated in the recycling of internalized proteins to the plasma membrane. However, the mechanism by which EHD1 mediates recycling and its relationship to Rab-family-controlled events has yet to be established. To investigate further the mode of EHD1 action, we sought to identify novel interacting partners. GST-EHD1 was used as bait to isolate a approximately 120-kDa species from bovine and murine brain cytosol, which was identified by mass spectrometry as the divalent Rab4/Rab5 effector Rabenosyn-5. We mapped the sites of interaction to the EH domain of EHD1, and the first two of five NPF motifs of Rabenosyn-5. Immunofluorescence microscopy studies revealed that EHD1 and Rabenosyn-5 partially colocalize to vesicular and tubular structures in vivo. To address the functional roles of EHD1 and Rabenosyn-5, we first demonstrated that RNA interference (RNAi) dramatically reduced the level of expression of each protein, either individually or in combination. Depletion of either EHD1 or Rabenosyn-5 delayed the recycling of transferrin and major histocompatibility complex class I to the plasma membrane. However, whereas depletion of EHD1 caused the accumulation of internalized cargo in a compact juxtanuclear compartment, Rabenosyn-5-RNAi caused its retention within a dispersed peripheral compartment. Simultaneous RNAi depletion of both proteins resulted in a similar phenotype to that observed with Rabenosyn-5-RNAi alone, suggesting that Rabenosyn-5 acts before EHD1 in the regulation of endocytic recycling. Our studies suggest that Rabenosyn-5 and EHD1 act sequentially in the transport of proteins from early endosomes to the endosomal recycling compartment and back to the plasma membrane.

Amino Acid Motifs↗

PubMed2006

The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

Animals↗

PubMed2001

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).

Alleles↗

PubMed2006

Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog.

RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid-mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4- or Nanog-targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes Cdx2, Hand1, and PL-1, with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes GATA4, GATA6, and laminin B1, with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose-dependent manner to influence cell fate.

Animals↗

PubMed2006

MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula.

In the model legume Medicago truncatula, we identified a new transcription factor of the CCAAT-binding family, MtHAP2-1, for which RNA interference (RNAi) and in situ hybridization experiments indicate a key role during nodule development, possibly by controlling nodule meristem function. We could also show that MtHAP2-1 is regulated by microRNA169, whose overexpression leads to the same nodule developmental block as MtHAP2-1 RNAi constructs. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR169-resistant MtHAP2-1 nodules strongly suggest, in addition, that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is essential for the differentiation of nodule cells.

Base Sequence↗

PubMed2004

Construction and transfection of PCR products expressing siRNAs or shRNAs in mammalian cells.

In mammalian cells, the RNA interference (RNAi) effect has been observed through expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evaluation of small interfering RNAs (siRNA) expression units in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells for functional testing. This method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy.

Animals↗

PubMed2005

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Cell Line, Tumor↗

PubMed2006

A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium.

For the application of RNA interference (RNAi) in vivo the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle. Therefore, delivery technologies need to be established for the systemic application of RNAi in vivo. Here we report uptake, biodistribution and in vivo efficacy of siRNA molecules formulated into siRNA-lipoplexes. The applied formulation is based on complex formation of positively charged liposomes, a mixture of cationic and fusogenic lipids complexed with the negatively charged siRNA. We determined by fluorescence microscopy the temporal and spatial distribution of fluorescently labeled siRNA-lipoplexes, the body clearance and endothelial cell type specific uptake after single intravenous injection. Furthermore, by using siRNA molecules for targeting endothelia-specifically expressed genes, such as CD31 and Tie2, we were able to demonstrate downregulation of the corresponding mRNA and protein in vivo. Taken together, we show the applicability of this non-viral delivery technology for inducing RNAi in the vasculature of mice after systemic application.

Animals↗

PubMed2004

Applications of nucleic acid technology in the CNS.

This review examines applications of nucleic acid technology in the form of catalytic nucleic acids (ribozymes and DNAzymes) and RNA interference (RNAi) in the CNS. The basic mechanism of catalytic nucleic acids and RNAi is reviewed, and potentials and problems highlighted. Recent advances in chemical modifications and delivery techniques are summarized. Applications in the CNS, including their use in primary neuronal cells, organotypic slice culture and the brain in vivo are further discussed.

Animals↗

PubMed2006

A novel guanine nucleotide exchange factor MyoGEF is required for cytokinesis.

The cleavage furrow is created by an actomyosin contractile ring that is regulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotide exchange factors (GEFs) are positive regulators of the small GTPase proteins and have been implicated as important factors in regulating cytokinesis. However, it is still unclear how GEFs regulate the contractile ring during cytokinesis in mammalian cells. Here we report that a novel GEF, which is termed MyoGEF (myosin-interacting GEF), interacts with non-muscle myosin II and exhibits activity toward RhoA. MyoGEF and non-muscle myosin II colocalize to the cleavage furrow in early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNA interference (RNAi) results in the formation of multinucleated cells. These results suggest that MyoGEF, RhoA, and non-muscle myosin II act as a functional unit at the cleavage furrow to advance furrow ingression during cytokinesis.

Animals↗

PubMed2006

The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled.

Dishevelled (Dvl) transduces signals from the Wnt receptor, Frizzled, to downstream components, leading to the stabilization of beta-catenin and subsequent activation of the transcription factor T cell factor (TCF) and/or lymphoid enchancer factor (LEF). However, the mechanism of Dvl action remains unclear. Here, we report that nucleoredoxin (NRX), a thioredoxin (TRX) family protein, interacts with Dvl. Overexpression of NRX selectively suppresses the Wnt-beta-catenin pathway and ablation of NRX by RNA-interference (RNAi) results in activation of TCF, accelerated cell proliferation and enhancement of oncogenicity through cooperation with mitogen-activated extracellular signal regulated kinase kinase (MEK) or Ras. We find that cells respond to H(2)O(2) stimulation by activating TCF. Redox-dependent activation of the Wnt-beta-catenin pathway occurs independently of extracellular Wnts and is impaired by RNAi of NRX . In addition, association between Dvl and NRX is inhibited by H(2)O(2) treatment. These data suggest a relationship between the Wnt-beta-catenin pathway and redox signalling through redox-sensitive association of NRX with Dvl.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Autoimmune targeting of key components of RNA interference.

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

Animals↗

PubMed2005

Functional genomic analysis of the Wnt-wingless signaling pathway.

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.

Animals↗

PubMed2005

The noncoding RNA taurine upregulated gene 1 is required for differentiation of the murine retina.

BACKGROUND: With the advent of genome-wide analyses, it is becoming evident that a large number of noncoding RNAs (ncRNAs) are expressed in vertebrates. However, of the thousands of ncRNAs identified, the functions of relatively few have been established. RESULTS: In a screen for genes upregulated by taurine in developing retinal cells, we identified a gene that appears to be a ncRNA. Taurine Upregulated Gene 1 (TUG1) is a spliced, polyadenylated RNA that does not encode any open reading frame greater than 82 amino acids in its full-length, 6.7 kilobase (kb) RNA sequence. Analyses of Northern blots and in situ hybridization revealed that TUG1 is expressed in the developing retina and brain, as well as in adult tissues. In the newborn retina, knockdown of TUG1 with RNA interference (RNAi) resulted in malformed or nonexistent outer segments of transfected photoreceptors. Immunofluorescent staining and microarray analyses suggested that this loss of proper photoreceptor differentiation is a result of the disregulation of photoreceptor gene expression. CONCLUSIONS: A function for a newly identified ncRNA, TUG1, has been established. TUG1 is necessary for the proper formation of photoreceptors in the developing rodent retina.

Amino Acid Sequence↗

PubMed2005

RNA interference during spermatogenesis in mice.

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.

Animals↗

PubMed2004

Molecular medicine for the brain: silencing of disease genes with RNA interference.

The recent discovery of RNA interference (RNAi) has revolutionised biological research and now holds promise as a potential therapy for human diseases. Currently untreatable neurological diseases are especially attractive targets. Scientists have already succeeded in using RNAi to suppress dominant disease genes in vitro; in some cases, this suppression has been allele-specific, silencing the disease-causing allele while maintaining expression of the normal allele. The challenge now is to bring this powerful technology in vivo to animal models to suppress disease genes and correct disease phenotypes. In the confrontation of this challenge, research should benefit from recent advances in viral and non-viral delivery of therapy to the brain.

Animals↗

PubMed2006

STAT6 specific shRNA inhibits proliferation and induces apoptosis in colon cancer HT-29 cells.

RNA interference (RNAi) has been employed as an effective tool for studying gene functions. Here, we have demonstrated that different short hairpin RNAs (shRNAs) can specifically knock-down the transcription of STAT6, resulting in the inhibition of cell proliferation, G1/S phase arrest and apoptotic death in colon cancer HT-29 cells. These findings suggest that Stat6 signaling pathway plays a role in the regulation of cell proliferation and apoptosis in colon cancer cells, which promises further investigation for targeted cancer therapy.

Apoptosis↗

PubMed2004

Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB.

Mx proteins form a subfamily of the dynamin-like GTPases, which have well established roles in cellular trafficking. Some Mx proteins (e.g., human MxA) have antiviral activity and are tightly regulated by type I IFNs. Others (e.g., human MxB) lack antiviral activity and are thought to have normal cellular functions that remain undefined. Consistent with this hypothesis, we report that MxB is expressed without IFN treatment. MxB seems to be exclusively extranuclear and is concentrated at the cytoplasmic face of nuclear pores, suggesting a role in their regulation. We find that expression of dominant negative (GTPase-defective) MxB mutants efficiently blocks nuclear import and causes a delay in G(1)/S cell-cycle progression. MxB depletion using RNA interference (RNAi) leads to a similar cell-cycle defect but does not block nuclear import. MxB therefore seems not to be required for nuclear import per se but may instead regulate its efficiency and/or kinetics. These studies indicate an unexpected role for a dynamin-like protein in nucleocytoplasmic trafficking and suggest that a related function might be usurped by its antiviral relatives.

Active Transport, Cell Nucleus↗