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1.
RNA-mediated interference (RNAi) has emerged recently as one of the most powerful functional genomics tools. RNAi has been
particularly effective in the nematode worm C. elegans where RNAi has been used to analyse the loss-of-function phenotypes of almost all predicted genes. In this review, we illustrate
how RNAi has been used to analyse gene function in C. elegans as well as pointing to some future directions for using RNAi to examine genetic interactions in a systematic manner. 相似文献
2.
Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division
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Mirko Theis Mikolaj Slabicki Magno Junqueira Maciej Paszkowski‐Rogacz Jana Sontheimer Anne‐Kristine Heninger Timo Glatter Kristi Kruusmaa Ina Poser Anthony A Hyman M Teresa Pisabarro Matthias Gstaiger Rudolf Aebersold Andrej Shevchenko Frank Buchholz 《The EMBO journal》2009,28(10):1453-1465
Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses. 相似文献
3.
The Rice OsRad21-4, an Orthologue of Yeast Rec8 Protein,is Required for Efficient Meiosis 总被引:9,自引:0,他引:9
In yeast, Rad21/Scc1 and its meiotic variant Rec8 are key players in the establishment and subsequent dissolution of sister
chromatid cohesion for mitosis and meiosis, respectively, which are essential for chromosome segregation. Unlike yeast, our
identification revealed that the rice genome has 4 RAD21-like genes that share lower than 21% identity at polypeptide levels, and each is present as a single copy in this genome.
Here we describe our analysis of the function of OsRAD21-4 by RNAi. Western blot analyses indicated that the protein was most abundant in young flowers and less in leaves and buds
but absent in roots. In flowers, the expression was further defined to premeiotic pollen mother cells (PMCs) and meiotic PMCs
of anthers. Meiotic chromosome behaviors were monitored from male meiocytes of OsRAD21-4-deficient lines mediated by RNAi.
The male meiocytes showed multiple aberrant events at meiotic prophase I, including over-condensation of chromosomes, precocious
segregation of homologues and chromosome fragmentation. Fluorescence in situ hybridization experiments revealed that the deficient lines were defective in homologous pairing and cohesion at sister chromatid
arms. These defects resulted in unequal chromosome segregation and aberrant spore generation. These observations suggest that
OsRad21-4 is essential for efficient meiosis. 相似文献
4.
Timmons LD 《Journal of bioenergetics and biomembranes》2007,39(5-6):459-463
RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded
RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach
using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants,
we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter
genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their
roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles
for some transporter genes in endogenous RNAi-related processes. 相似文献
5.
Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference
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Somma MP Ceprani F Bucciarelli E Naim V De Arcangelis V Piergentili R Palena A Ciapponi L Giansanti MG Pellacani C Petrucci R Cenci G Vernì F Fasulo B Goldberg ML Di Cunto F Gatti M 《PLoS genetics》2008,4(7):e1000126
6.
Jeehee Kim Aurora Badaloni Torsten Willert Ursula Zimber-Strobl Ralf Kühn Wolfgang Wurst Matthias Kieslinger 《PloS one》2013,8(11)
Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo. 相似文献
7.
Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis. 相似文献
8.
Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism. 相似文献
9.
In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity
genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little
is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway (Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in
these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became
highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these
small spherical embryos. On the other hand, RNAi analysis of a negative regulator (Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar
larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en–wg–hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove
formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect. 相似文献
10.
A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in
a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function.
However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods,
we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled
double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated
into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer
dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest
that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting
points for the development of functional protocols in nonmodel organisms. 相似文献
11.
《Cell cycle (Georgetown, Tex.)》2013,12(17):3591-3601
Greatwall (Gwl) was originally discovered in Drosophila as an essential kinase for correct chromosome condensation and mitotic progression. In Xenopus, Gwl may influence the positive-feedback loop that directs cyclin B1-Cdk1 activation and the mitotic state by inhibiting the phosphatase PP2A. Here, we describe the human orthologue of Gwl called microtubule-associated serine/threonine kinase-like (MASTL). We found that MASTL localizes to the nucleus in interphase and re-localizes in part to centrosomes in mitosis, when it is active. Cells strongly depleted of MASTL by RNAi delay in G2 phase and reveal slow chromosome condensation. MASTL RNAi cells that enter and progress through mitosis often fail to completely separate their sister chromatids in anaphase. This causes chromatin to be trapped in the cleavage furrow, which may lead to formation of 4N G1 cells by cytokinesis failure. Further, our experiments indicate that MASTL supports the phosphorylation state of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C. Cyclin B1 destruction is incomplete when mitotic cells that are strongly depleted of MASTL exit mitosis. We propose that MASTL enhances cyclin B1-Cdk1-dependent mitotic phosphorylation-events, directing mitotic entry, anaphase and cytokinesis in human cells. 相似文献
12.
利用RNAi技术大规模分析基因功能的研究 总被引:1,自引:0,他引:1
将双链RNA导入细胞内会干扰与之同源的基因的表达 ,使生物体产生相应的功能缺陷表型 ,这种作用称为RNAi(RNAinterference)。针对人类、植物、微生物等大规模基因组测序后所面临的功能鉴定难题 ,着重探讨了RNAi的机制及其研究进展。复合物RISC和酶Dicer的发现揭示了RNAi导致同源基因沉默的作用机理。通过使用RNAi这种反向遗传学工具可使生物体产生相应的功能缺陷表型 ,从而确定未知基因的功能。因此RNAi对大规模分析动、植物基因功能的研究具有重要的作用。 相似文献
13.
14.
Zhiqian Li Baosheng Zeng Lin Ling Jun Xu Lang You Abu F.M. Aslam Anjiang Tan Yongping Huang 《International journal of biological sciences》2015,11(2):176-185
RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general. 相似文献
15.
16.
《遗传学报》2015,(6)
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs(sg RNAs).Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing(HTS).We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies. 相似文献
17.
18.
The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells
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Annemarthe G van der Veen Pierre V Maillard Jan Marten Schmidt Sonia A Lee Safia Deddouche‐Grass Annabel Borg Svend Kjær Ambrosius P Snijders Caetano Reis e Sousa 《The EMBO journal》2018,37(4)
In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins. 相似文献
19.
Zhen Wang Changbin Chen Yunyuan Xu Rongxi Jiang Ye Han Zhihong Xu Kang Chong 《Plant Molecular Biology Reporter》2004,22(4):409-417
In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide
range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by
an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency
of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin
promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector,
only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct,
which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed
the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately
85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms
facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing
a rice gene.
These authors contributed equally to this work. 相似文献