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PubMed2006

Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane.

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.

Animals↗

PubMed2006

The late pollen actins are essential for normal male and female development in Arabidopsis.

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3'UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.

Actins↗

PubMed2006

HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing.

We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the 'dicer-related-helicase' from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF- cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF- cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.

Amino Acid Sequence↗

PubMed2004

Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons.

The neuropathological hallmark of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra pars compacta, presumably mediated by apoptosis. The homeobox transcription factors engrailed 1 and engrailed 2 are expressed by this neuronal population from early in development to adulthood. Despite a large mid-hindbrain deletion in double mutants null for both genes, mesencephalic dopaminergic (mDA) neurons are induced, become postmitotic and acquire their neurotransmitter phenotype. However, at birth, no mDA neurons are left. We show that the entire population of these neurons is lost by E14 in the mutant animals, earlier than in any other described genetic model system for Parkinson's disease. This disappearance is caused by apoptosis revealed by the presence of activated caspase 3 in the dying tyrosine hydroxylase-positive mutant cells. Furthermore, using in vitro cell mixing experiments and RNA interference on primary cell culture of ventral midbrain we were able to show that the demise of mDA neurons in the mutant mice is due to a cell-autonomously requirement of the engrailed genes and not a result of the missing mid-hindbrain tissue. Gene silencing in the postmitotic neurons by RNA interference activates caspase 3 and induces apoptosis in less than 24 hours. This rapid induction of cell death in mDA neurons suggests that the engrailed genes participate directly in the regulation of apoptosis, a proposed mechanism for Parkinson's disease.

Animals↗

PubMed2006

A functional genomics analysis of the B56 isoforms of Drosophila protein phosphatase 2A.

Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B') but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila.

Animals↗

PubMed2005

Arabidopsis epigenetics: when RNA meets chromatin.

Recent work in plants and other eukaryotes has uncovered a major role for RNA interference in silent chromatin formation. The heritability of the silent state through multiple cell division cycles and, in some instances, through meiosis is assured by epigenetic marks. In plants, transposable elements and transgenes provide striking examples of the stable inheritance of repressed states, and are characterized by dense DNA methylation and heterochromatin histone modifications. Arabidopsis is a useful higher eukaryotes model with which to explore the crossroads between silent chromatin and RNA interference both during development and in the genome-wide control of repeat elements.

Arabidopsis↗

PubMed2005

RNA interference-mediated silencing of the syntaxin 5 gene induces Golgi fragmentation but capable of transporting vesicles.

It has been suggested that syntaxin 5 (Syx5) participates in vesicular transport. We examined the effects of Syx5 down-regulation on the morphology of the Golgi apparatus and the transport of vesicles in mammalian cells. Knockdown of the Syx5 gene resulted in Golgi fragmentation without changing the level of endoplasmic reticulum (ER)-resident proteins, other Golgi-SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors), and coatmer proteins. Strikingly, a major decrease in Syx5 expression barely affected the anterograde transport of vesicular stomatitis virus G (VSVG) protein to the plasma membrane. These results suggest that Syx5 is required for the maintenance of the Golgi structures but may not play a major role in the transport of vesicles carrying VSVG between the ER and the Golgi compartment.

Amino Acid Sequence↗

PubMed2005

RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3.

The recent increase in tropospheric ozone (O(3)) concentrations promotes additional oxidative stress through the production of reactive oxygen species (ROS) in plant tissues, resulting in the activation of genes whose products enable the stressed cells to retain their integrity and function. This response is made possible by an integration of highly regulated signaling networks that mediate the perception of, and response to, this oxidative assault. In Arabidopsis thaliana, ROS-induced signaling has been shown to flow through a protein phosphorylation cascade involving the mitogen-activated protein kinases (MAPKs) AtMPK3 (MPK3) and AtMPK6 (MPK6). We found that RNAi-mediated silencing of MPK6 renders the plant more sensitive to ozone, as determined by visible leaf damage. The MPK6-RNAi genotype also displayed a more intense and prolonged activation of MPK3 compared to that of WT plants. An MPK3 loss-of-function genotype is similarly very sensitive to ozone, and displays an abnormally prolonged MPK6 activation profile, suggesting reciprocity in regulation between these two MAPKs.

Arabidopsis↗

PubMed2005

The cellular HIV-1 Rev cofactor hRIP is required for viral replication.

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after reintroduction of hRIP protein. Taken together, our results indicate that hRIP is an essential cellular cofactor for Rev function and HIV-1 replication. Because hRIP is not required for cell viability, it may be an attractive target for the development of new antiviral strategies.

Gene Products, rev↗

PubMed2006

[Preparation of small interfering RNA expression cassette based on PCR technique].

OBJECTIVE: To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference. METHODS: The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference. RESULTS: The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection. CONCLUSION: The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing↗

PubMedDate not supplied

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects.

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

Animals↗

PubMed2002

Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies.

Alternative Splicing↗

PubMed2005

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Animals↗

PubMed2005

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1.

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.

Butadienes↗

PubMed2004

Mina53 as a potential prognostic factor for esophageal squamous cell carcinoma.

PURPOSE: We previously identified mina53, a novel Myc target gene. Here we investigated whether mina53 is related to esophageal squamous cell carcinoma (ESCC), a disease with poor prognosis. EXPERIMENTAL DESIGN: Mina53 expression was suppressed in ESCC cell lines by a RNA interference method to investigate whether Mina53 is involved in cell proliferation. Expression of Mina53 was investigated by Western blotting in tissue sections from patients with ESCC. Immunohistochemical analysis of Mina53 was carried out and compared with that using anti-Ki-67 antibody. Finally, the level of Mina53 expression was compared with the length of survival of patients with ESCC. RESULTS: Reduction of mina53 expression by RNA interference suppressed cell proliferation in ESCC cell lines. Western blot analysis of surgically resected ESCC specimens indicated that the expression of Mina53 in tumors was increased compared with that in adjacent nonneoplastic tissues in all four specimens examined. When formalin-fixed specimens from 52 patients with ESCC were stained immunohistochemically, it was found that Mina53 was highly expressed in 83% of specimens. Anti-Mina53 antibody stained tumors more efficiently than antibody against Ki-67, a cell proliferation biomarker, in some cancer specimens. Patients with high expression of Mina53 had shorter survival periods, whereas the expression level of Ki-67 in ESCC showed no relationship to patient outcome. CONCLUSIONS: Taken together, our results indicate that expression of Mina53 is a characteristic feature of ESCC and suggest that immunostaining by anti-Mina53 antibody may be useful as a potential prognostic indicator.

Blotting, Western↗

PubMed2005

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Autoantigens↗

PubMed1999

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.

Animals↗

PubMed2006

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation.

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.

Animals↗