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PubMed2003

Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

Animals↗
PubMed2002

Gene silencing using micro-RNA designed hairpins.

During RNA interference (RNAi), long dsRNA is processed to approximately 21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are approximately 21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.

Blotting, Northern↗
PubMed2002

The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis.

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7alpha and CeGLC-7beta, abolishes the restricted localization pattern of AIR-2. In Ceglc-7alpha/beta(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7alpha/beta(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7alpha/beta, directly or indirectly, antagonize AIR-2 activity.

Anaphase↗
PubMed2003

The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.

Anaphase↗
PubMed2004

Communication between parental and developing genomes during tetrahymena nuclear differentiation is likely mediated by homologous RNAs.

Approximately 6000 DNA elements, totaling nearly 15 Mb, are coordinately excised from the developing somatic genome of Tetrahymena thermophila. An RNA interference (RNAi)-related mechanism has been implicated in the targeting of these germline-limited sequences for chromatin modification and subsequent DNA rearrangement. The excision of individual DNA segments can be inhibited if the homologous sequence is placed within the parental somatic nucleus, indicating that communication occurs between the parental and developing genomes. To determine how the DNA content of one nucleus is communicated to the other, we assessed DNA rearrangement occurring in wild-type cells that were mated to cells that contained the normally germline-limited M element within their somatic nuclei. M-element rearrangement was blocked in the wild-type cell even when no genetic exchange occurred between mating partners, a finding that is inconsistent with any genetic imprinting models. This inhibition by the parental somatic nucleus was rapidly established between 5 and 6 hr of conjugation, near or shortly after the time that zygotic nuclei are formed. M-element small RNAs (sRNAs) that are believed to direct its rearrangement were found to rapidly accumulate during the first few hours of conjugation before stabilizing to a low, steady-state level. The period between 5 and 6 hr during which sRNA levels stabilize correlates with the time after which the parental genome can block DNA rearrangement. These data lead us to suggest that homologous sRNAs serve as mediators to communicate sequence-specific information between the parental and developing genomes, thereby regulating genome-wide DNA rearrangement, and that these sRNAs can be effectively compared to the somatic genome of both parents.

Animals↗
PubMed2005

A partial down-regulation of WASP is sufficient to inhibit podosome formation in dendritic cells.

The Wiskott Aldrich syndrome protein (WASP) is a hematopoietic-specific cytoskeletal regulator that is necessary for induction of normal immunity. In the context of effective gene therapy for WAS, cellular models of human WASP deficiency are important for definition of the threshold of protein expression required for optimal activity. Using lentiviral vector-mediated RNA interference (RNAi), we were able to down-regulate the levels of human WASP in cell lines and primary cells. In dendritic cells (DC), RNAi-induced WASP deficiency did not impair phenotypic maturation but perturbed cytoskeletal organization. As a result, podosomes, which are actin-rich structures present in immature adherent DC, were formed less efficiently and motility was disturbed. Overall, treatment of cells with RNAi recapitulated the phenotype of cells derived from patients or animals with inactivating mutations of the WAS gene. Interestingly, reduction of the levels of WASP to about 60% of normal was sufficient to inhibit the formation of podosomes in DC, implying that this cell type requires near-normal levels of WASP to sustain physiological cytoskeleton-dependent activities.

Cell Movement↗
PubMed2002

U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.

Animals↗
PubMed2006

Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs.

RNA interference (RNAi) is a well-conserved, ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. The endogenous small RNAs, called microRNAs, are processed from hairpin precursors and regulate important genes involved in cell death, differentiation, and development. RNAi also protects the genome from invading genetic elements, encoded by transposons and viruses. When small double-stranded RNAs, called small interfering (si)RNAs, are introduced into cells, they bind to the endogenous RNAi machinery to disrupt the expression of mRNAs containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. Pilot siRNA clinical studies began just three years after the discovery that RNAi works in mammalian cells. This review discusses recent progress and obstacles to using siRNAs as small molecule drugs.

Animals↗
PubMed2002

The SIN3/RPD3 deacetylase complex is essential for G(2) phase cell cycle progression and regulation of SMRTER corepressor levels.

The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. Here we show that the SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G(2) phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G(2). Thus, the role of the SIN3/RPD3 complex in G(2) phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G(2) phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G(2) phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes.

Acetylation↗
PubMed2006

MEKK4 signaling regulates filamin expression and neuronal migration.

Periventricular heterotopia (PVH) is a congenital malformation of human cerebral cortex frequently associated with Filamin-A (FLN-A) mutations but the pathogenetic mechanisms remain unclear. Here, we show that the MEKK4 (MAP3K4) pathway is involved in Fln-A regulation and PVH formation. MEKK4(-/-) mice developed PVH associated with breaches in the neuroependymal lining which were largely comprised of neurons that failed to reach the cortical plate. RNA interference (RNAi) targeting MEKK4 also impaired neuronal migration. Expression of Fln was elevated in MEKK4(-/-) forebrain, most notably near sites of failed neuronal migration. Importantly, recombinant MKK4 protein precipitated a complex containing MEKK4 and Fln-A, and MKK4 mediated signaling between MEKK4 and Fln-A, suggesting that MKK4 may bridge these molecules during development. Finally, we showed that wild-type FLN-A overexpression inhibited neuronal migration. Collectively, our results demonstrate a link between MEKK4 and Fln-A that impacts neuronal migration initiation and provides insight into the pathogenesis of human PVH.

Animals↗
PubMed2001

Role for a bidentate ribonuclease in the initiation step of RNA interference.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Animals↗
PubMed2005

Restriction of feline immunodeficiency virus by Ref1, Lv1, and primate TRIM5alpha proteins.

The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5alpha proteins and performed RNA interference (RNAi) against endogenous TRIM5alpha. We find that expression of rhesus or human TRIM5alpha proteins in feline cells restricts FIV, impairing pseudotyped vector transduction and viral replication, but rhesus TRIM5alpha is more restricting than human TRIM5alpha. Notably, however, canine cells did not support restriction by human TRIM5alpha and supported minimal restriction by rhesus TRIM5alpha, suggesting that these proteins may not function autonomously or that a canine factor interferes. Stable RNAi knockdown of endogenous rhesus TRIM5alpha resulted in marked increases in FIV and HIV-1 infectivities while having no effect on NB-MLV. A panel of nonprimate cell lines varied widely in susceptibility to lentiviral vector transduction, but normalized FIV and HIV-1 vectors varied concordantly. In contrast, in human and monkey cells, relative restriction of FIV compared to HIV-1 varied from none to substantial, with the greatest relative infectivity deficit for FIV vectors observed in human T-cell lines. Endogenous and introduced TRIM5alpha restrictions of FIV could be titrated by coinfections with FIV, HIV-1, or EIAV virus-like particles. Arsenic trioxide had complex and TRIM5alpha-independent enhancing effects on lentiviral but not NB-MLV infection. Implications for human gene therapy are discussed.

Animals↗
PubMed2005

CCT chaperonin complex is required for the biogenesis of functional Plk1.

Experiments from several different organisms have demonstrated that polo-like kinases are involved in many aspects of mitosis and cytokinesis. Here, we provide evidence to show that Plk1 associates with chaperonin-containing TCP1 complex (CCT) both in vitro and in vivo. Silencing of CCT by use of RNA interference (RNAi) in mammalian cells inhibits cell proliferation, decreases cell viability, causes cell cycle arrest with 4N DNA content, and leads to apoptosis. Depletion of CCT in well-synchronized HeLa cells causes cell cycle arrest at G(2), as demonstrated by a low mitotic index and Cdc2 activity. Complete depletion of Plk1 in well-synchronized cells also leads to G(2) block, suggesting that misfolded Plk1 might be responsible for the failure of CCT-depleted cells to enter mitosis. Moreover, partial depletion of CCT or Plk1 leads to mitotic arrest. Finally, the CCT-depleted cells reenter the cell cycle upon reintroduction of the purified constitutively active form of Plk1, indicating that Plk1 might be a CCT substrate.

Animals↗
PubMed2006

Off-target effects by siRNA can induce toxic phenotype.

Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.

Base Sequence↗
PubMed2004

siRNA-directed silencing of transgene expressed in cultured insect cells.

RNA interference (RNAi) has emerged as a powerful tool to rapidly analyze gene functions in a wide variety of eukaryotic organisms as well as in cultured cell lines. We demonstrate here that RNAi can be applied to study the function of a transgene expressed in an insect cell line (Spodoptera frugiperda, Sf21). The aminopeptidase N gene (apn) targeted for silencing in the present study was isolated from the midgut of Spodoptera litura larvae and expressed in Sf21 cells using baculovirus expression system. The recombinant APN protein expressed at the surface of Sf21 cells was shown to interact with insecticidal crystal protein, Cry1C, by in vitro experiments. The exogenous addition/transfection of APN dsRNA or siRNA in the cultured cells resulted in partial/complete inhibition of expression of apn leading to the loss of toxin binding to the transgene expressing cells. These experiments highlighted the usefulness of RNAi as a tool to study the function of an expressed transgene in insect cell line and to study the specificity of receptor-ligand interaction.

Animals↗
PubMed2005

Multigenic families and proteomics: extended protein characterization as a tool for paralog gene identification.

In classical proteomic studies, the searches in protein databases lead mostly to the identification of protein functions by homology due to the non-exhaustiveness of the protein databases. The quality of the identification depends on the studied organism, its complexity and its representation in the protein databases. Nevertheless, this basic function identification is insufficient for certain applications namely for the development of RNA-based gene-silencing strategies, commonly termed RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants, that require an unambiguous identification of the targeted gene sequence. A PTGS strategy was considered in the study of the infection of Oryza sativa by the Rice Yellow Mottle Virus (RYMV). It is suspected that the RYMV recruits host proteins after its entry into plant cells to form a complex facilitating virus multiplication and spreading. The protein partners of this complex were identified by a classical proteomic approach, nano liquid chromatography tandem mass spectrometry. Among the identified proteins, several were retained for a PTGS strategy. Nevertheless most of the protein candidates appear to be members of multigenic families for which all paralog genes are not present in protein databases. Thus the identification of the real expressed paralog gene with classical protein database searches is impossible. Consequently, as the genome contains all genes and thus all paralog genes, a whole genome search strategy was developed to determine the specific expressed paralog gene. With this approach, the identification of peptides matching only a single gene, called discriminant peptides, allows definitive proof of the expression of this identified gene. This strategy has several requirements: (i) a genome completely sequenced and accessible; (ii) high protein sequence coverage. In the present work, through three examples, we report and validate for the first time a genome database search strategy to specifically identify paralog genes belonging to multigenic families expressed under specific conditions.

Chaperonin 60↗
PubMed2006

Genetic interactions among cortical malformation genes that influence susceptibility to convulsions in C. elegans.

Epilepsy is estimated to affect 1-2% of the world population, yet remains poorly understood at a molecular level. We have previously established the roundworm Caenorhabditis elegans as a model for investigating genetic susceptibilities to seizure-like convulsions in vivo. Here we investigate the behavioral consequences of decreasing the activity of nematode gene homologs within the LIS1 pathway that are associated with a human cortical malformation termed lissencephaly. Bioinformatic analysis revealed the nud-2 gene, encoding the worm homolog of mammalian effectors of LIS1, termed NDE1 and NDEL1. Phenotypic analysis of animals targeted by RNA interference (RNAi) was performed using a pentylenetetrazole (PTZ) exposure paradigm to induce convulsions. Worms depleted for LIS1 pathway components (NUD-1, NUD-2, DHC-1, CDK-5, and CDKA-1) exhibited significant convulsions following PTZ and RNAi treatment. Strains harboring fluorescent markers for GABAergic neuronal architecture and synaptic vesicle trafficking were employed to discern putative mechanisms accounting for observed convulsion behaviors. We found that depletion of LIS1 pathway components resulted in defective GABA synaptic vesicle trafficking. We also utilized combinations of specific genetic backgrounds to create a sensitized state for convulsion susceptibility and discovered that convulsion effects were significantly enhanced when LIS-1 and other pathway components were compromised within the same animals. Thus, interactions among gene products with LIS-1 may mediate intrinsic thresholds of neuronal synchrony.

Animals↗
PubMed2002

Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis.

Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. Here, we present the identification of Drosophila Myt1 (dMyt1) kinase and examine the relationship of Myt1 and Wee1 activities in the context of cdc2 phosphorylation. dMyt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells resulted in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) resulted in an increased rate of proliferation. In addition, loss of dMyt1 alone or in combination with Drosophila Wee1 (dWee1) resulted in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of dMyt1 alone resulted in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of cdc2 on the tyrosine-15 (Tyr-15) residue was only observed when both dMyt1 and dWee1 expression was reduced via RNAi and not by Wee1 alone. Most strikingly, in the absence of dMyt1, Golgi fragmentation during mitosis was incomplete. Our findings suggest that dMyt1 and dWee1 have distinct roles in the regulation of cdc2 phosphorylation and the regulation of mitotic events.

Amino Acid Sequence↗