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PubMed2004

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis.

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.

Amino Acid Sequence↗
PubMed2006

[Study of effects of HBV X gene and As2O3 on expression and activity of p53 in HepG2 cells with shRNA].

OBJECTIVE: To delineate the effects of HBV X gene and of As(2)O(3) on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference (RNAi). METHODS: HepG2 cells and cells with stable expression of HBV X gene, HepG2-X, were treated with 2 micromol/L As(2)O(3), and the corresponding untreated cells were used as controls. Cell and nuclear lysates were extracted. Total level and the relative activity absorbance of p53 were detected by modified ELISA. HBV X gene sequence-specific shRNA expression vectors, Xi-S1 and Xi-S2, and sequence-unrelated control Xi-S3 were transfected into HepG2-X. The effect of As(2)O(3) on p53 expression and activity were retested. RESULTS: Total p53 level was up-regulated and its relative activity ratio was enhanced by As(2)O(3) in HepG2 and HepG2-X cells. The total p53 level induced by As(2)O(3) was further up-regulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA. CONCLUSION: As(2)O(3) could up-regulate p53 expression and enhance its activity. shRNA-mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity. HBV X expression could up-regulate p53 gene expression level induced by As(2)O(3), while it suppressed the activity of p53.

Apoptosis↗
PubMed2004

Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes.

Double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation in most eukaryotic organisms via a conserved pathway known as RNA interference (RNAi). Post-transcriptional gene silencing by RNAi is also connected with transcriptional silencing of cognate sequences. In plants, this transcriptional silencing is associated with sequence-specific DNA methylation. To address whether this mechanism operates in mammalian cells, we used bisulfite sequencing to analyze DNA in mouse oocytes constitutively expressing long dsRNA against the Mos gene. Our data show that long dsRNA induces efficient Mos mRNA knockdown but not CpG and non-CpG DNA methylation of the endogenous Mos sequence in oocytes and early embryos. These data demonstrate that dsRNA does not directly induce DNA methylation in the trans form of this sequence in these mammalian cells.

Animals↗
PubMed2005

Inhibition of SARS-CoV replication by siRNA.

Serious outbreaks of severe acute respiratory syndrome (SARS), caused by the newly discovered coronavirus SARS-CoV, occurred between late 2002 and early 2003 and there is an urgent need for effective antiviral agents. RNA interference in animals and post-transcriptional gene silencing plants is mediated by small double-stranded RNA molecules named small interfering RNA (siRNA). Recently, siRNA-induced RNA interference(RNAi) may provide a new approach to therapy for pathogenic viruses, e.g. HIV and HCV. In this study, the silencing potential of seven synthetic siRNAs against SARS-CoV leader, TRS, 3'-UTR and Spike coding sequence have been applied to explore the possibility for prevention of SARS-CoV infection. We demonstrate that siRNAs directed against Spike sequences and the 3'-UTR can inhibit the replication of SARS-CoV in Vero-E6 cells, and holds out promise for the development of an effective antiviral agent against SARS-CoV.

3' Untranslated Regions↗
PubMed2006

Heteromerization of innexin gap junction proteins regulates epithelial tissue organization in Drosophila.

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.

Amino Acid Sequence↗
PubMed2006

Functional role of aspartic proteinase cathepsin D in insect metamorphosis.

BACKGROUND: Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. RESULTS: Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. CONCLUSION: Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

Animals↗
PubMed2006

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability.

Investigations aimed at identifying regulators of nuclear architecture in Drosophila demonstrated that cells lacking H3K9 methylation and RNA interference (RNAi) pathway components displayed disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared with wild type. We also observed a substantial increase in extrachromosomal circular (ecc) repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 and ecc DNA formation is not induced by removal of cohesin. We conclude that the structural integrity and organization of repeated DNAs and nucleoli are regulated by the H3K9 methylation and RNAi pathways, and other regulators of heterochromatin-mediated silencing. In addition, repeated DNA stability involves suppression of non-homologous end joining (NHEJ) or other recombination pathways. These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.

Animals↗
PubMed2006

Improvements in siRNA properties mediated by 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA).

RNA interference (RNAi) has emerged recently as an efficient mechanism for specific gene silencing. Short double-stranded small interfering RNAs (siRNAs) are now widely used for cellular or drug target validation; however, their use for silencing clinically relevant genes in a therapeutic setting remains problematic because of their unfavourable metabolic stability and pharmacokinetic properties. To address some of these concerns, we have investigated the properties of siRNA modified with 2'-deoxy-2'-fluoro-beta-d-arabinonucleotide units (araF-N or FANA units). Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA. We also show that the incorporation of FANA units into siRNA duplexes increases activity and substantially enhances serum stability of the siRNA. A fully modified sense 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) strand when hybridized to an antisense RNA (i.e. FANA/RNA hybrid) was shown to be 4-fold more potent and had longer half-life in serum (approximately 6 h) compared with an unmodified siRNA (<15 min). While incorporation of FANA units is well tolerated throughout the sense strand of the duplex, modifications can also be included at the 5' or 3' ends of the antisense strand, in striking contrast to other commonly used chemical modifications. Taken together, these results offer preliminary evidence of the therapeutic potential of FANA modified siRNAs.

Arabinonucleotides↗
PubMed2005

Two mink parvoviruses use different cellular receptors for entry into CRFK cells.

Mink enteritis virus (MEV) and Aleutian mink disease parvovirus (ADV) are two mink parvoviruses that replicate permissively in Crandell feline kidney (CRFK) cells. We have used this cell model to examine if these two mink parvoviruses use the same cellular receptor. Whereas the cellular receptor for MEV is expected to be the transferrin receptor (TfR), the cellular receptor for ADV has not been clearly identified. We used short hairpin RNAs (shRNAs) produced from plasmids to trigger RNA interference (RNAi), specifically and effectively reducing TfR expression in CRFK cells. TfR expression was reduced to levels undetectable by immunofluorescence in the majority of cells. In viral infection assays, we show that TfR expression was necessary for MEV infection but was not required for ADV infection. Thus, our results demonstrate that TfR is the cellular receptor for MEV, but not the cellular receptor for ADV. The use of two different receptors by MEV and ADV to infect the same cell line is yet another difference between these two parvoviruses that may contribute to their unique pathogenesis in mink.

Aleutian Mink Disease Virus↗
PubMed2003

Modeling human peroxisome biogenesis disorders in the nematode Caenorhabditis elegans.

Peroxisomes are ubiquitous eukaryotic organelles. The proteins required for peroxisome biogenesis are called peroxins, and mutations in the peroxin genes cause the devastating human developmental syndromes called the peroxisome biogenesis disorders. Our interest is in elaborating the roles that peroxisomes play in Caenorhabditis elegans development, and in establishing an invertebrate model system for the human peroxisome biogenesis disorders. The genome of C. elegans encodes homologs of 11 of the 13 human peroxins. We disrupted five nematode peroxins using RNA interference (RNAi) and found that RNAi knockdown of each one causes an early larval arrest at the L1 stage. Using a green fluorescent protein reporter targeted to the peroxisome, we establish that peroxisomal import is impaired in prx-5(RNAi) nematodes. prx-5(RNAi) animals are blocked very early in the L1 stage and do not initiate normal postembryonic cell divisions, similar to starvation-arrested larvae. Cell and axonal migrations that normally occur during the L1 stage also appear blocked. We conclude that peroxisome function is required for C. elegans postembryonic development and that disruption of peroxisome assembly by prx-5(RNAi) prevents scheduled postembryonic cell divisions. Defects in the cellular localization of peroxisomal proteins and in development are shared features of human and nematode peroxisome biogenesis disorders. In setting up a C. elegans model of peroxisomal biogenesis disorders, we suggest that genetic screens for suppression of the Prx developmental block will facilitate identification of novel intervention strategies and may provide new insights into human disease pathogenesis.

Animals↗
PubMed2006

Mutual information-based feature selection in studying perturbation of dendritic structure caused by TSC2 inactivation.

In this study, the effect of protein Tuberous sclerosis 2 (TSC2) on the dendritic spine density and length was demonstrated by using TSC2-RNAinactivation. In addition, the role of rapamycin, an antagonist of the molecular target of rapamycin, in the morphological changes of spine caused by TSC2 silencing was investigated. The features were extracted from highresolution three-dimensional image stacks collected by two-photon laser scanning microscopy of green fluorescing pyramidal cells expressing TSC2-RNA interference (RNAi), or TSC2-RNAi and rapamycin treatment in rat hippocampal slice cultures. We proposed to apply the lognormal distribution method for feature extraction. The extracted features of three cases under investigation, namely, (1) green-fluorescent protein GFP vs TSC2-RNAi, (2) GFP vs TSC2-RNAi and rapamycin, and (3) TSC2-RNAi vs TSC2-RNAi and rapamycin, were analyzed by mutual information-based feature selection and evaluated by three classifiers, K-nearest neighbor, Perceptron, and two-layer neural networks. The results showed that both the spine density and length have significant morphological changes after TSC2-RNAi treatment. However, rapamycin treatment could reverse the effect of TSC2-RNAi on spine length but not on spine density. These results are consistent with the results reported in the scientific literature. Finally, we explored the application of pattern recognition method in a small sample with richer feature properties, namely bootstrap mutual information estimation and a mutual information- based feature selection method.

Algorithms↗
PubMed2005

The terminal phase of cytokinesis in the Caenorhabditis elegans early embryo requires protein glycosylation.

RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.

Amino Acid Sequence↗
PubMed2006

Flagellar motility is required for the viability of the bloodstream trypanosome.

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

Animals↗
PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗
PubMed2004

Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival.

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B'') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B'' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.

Animals↗
PubMed2006

RNA interference effectively inhibits mRNA accumulation and protein expression of hepatitis C virus core and E2 genes in human cells.

RNA interference (RNAi) has been widely used for the analysis of gene function and represents a new promising approach to develop effective antiviral drugs. In this study, several small interfering RNAs (siRNAs) corresponding to two structural genes (core and E2) of hepatitis C virus (HCV) were designed and in vitro transcribed to explore the possibility of silencing these two genes. The plasmids pEGFP-C and pEGFP-E2, which contain the EGFP reporter gene and the core or E2 gene as silencing targets, were co-transfected with siRNAs into HEK 293T cells. At various time points of post-transfection, core and E2 expression levels were detected by fluorescence microscopy, flow cytometry, Western blotting, and real-time quantitative PCR. The results showed that the mean fluorescence intensity, protein expression, and RNA transcripts of siRNAs transfected cells were significantly reduced. This may provide an approach for the development of novel prophylactic or therapeutic agents for HCV infection.

Blotting, Western↗
PubMed2001

Embryonic XMab21l2 expression is required for gastrulation and subsequent neural development.

Cell fate determining gene mab-21 regulates the proper establishment of neural cell fate and sensory organ identity in nematode. Mammalian homologs of mab-21 have also been implicated to play critical roles in mid-, hindbrain and craniofacial differentiation. We report here the isolation of a mab-21 homolog, XMab21l2, from Xenopus. We showed that its expression in Xenopus was initiated at gastrulation and prominent signal was detected in neurulating embryos at the neural tube, the optic tissue, the developing midbrain, and the pharyngeal pouches. We demonstrated by RNA interference (RNAi), together with other antisense approaches, that XMab21l2 expression is required for the completion of gastrulation and subsequent neural development.

Amino Acid Sequence↗
PubMed2005

A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells.

Discovered as a DNA repair protein, Ape1 has been associated with other functions, notably redox regulation of transcription factors (Ref1 activity). Because deletion of the mouse gene produces embryonic lethality and stable Ape1-deficient cell lines have not been reported, there has been uncertainty about a possible vital cellular function of Ape1. We addressed this issue by using RNA interference (RNAi) in several human cell types. Strong downregulation of Ape1 stopped cell proliferation and activated apoptosis, which was correlated with accumulation of abasic DNA damage. These effects were reversed by expression of yeast Apn1 protein, which is structurally unrelated to Ape1 but shares enzymatic activity in repair of abasic sites (AP endonuclease). Because Apn1 would lack Ref1 activity or the protein interactions of Ape1, we conclude that the AP endonuclease activity is essential for cellular viability. Accumulation of abasic DNA damage from intrinsic sources appears sufficient to trigger cell death when Ape1-mediated repair is deficient.

Animals↗