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Genome-scale RNAi profiling of cell division in human tissue culture cells

ABSTRACT: Cell division is a fundamental process for multicellula organisation. Here we report a genome-scale RNAi screen in HeLa cells designed to identify human genes important for cell division. We utilized a library of endoribonuclease-prepared siRNAs (esiRNAs) for gene-silencing and used DNA content analysis to uncover genes that induced cell cycle arrest or altered ploidy upon knockdown. Rigorous validation and secondary assays were preformed to generate a nine-parameter fingerprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by describing novel functions to genes in mitosis and cytokinesis. In particular, we identified two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis in mammalian cells. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin. Keywords: time course

ORGANISM(S): Homo sapiens

PROVIDER: GSE9176 | GEO | 2007/11/01

SECONDARY ACCESSION(S): PRJNA102741

REPOSITORIES: GEO

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Genome-scale RNAi profiling of cell division in human tissue culture cells

Project description:Cell division is a fundamental process for multicellula organisation. Here we report a genome-scale RNAi screen in HeLa cells designed to identify human genes important for cell division. We utilized a library of endoribonuclease-prepared siRNAs (esiRNAs) for gene-silencing and used DNA content analysis to uncover genes that induced cell cycle arrest or altered ploidy upon knockdown. Rigorous validation and secondary assays were preformed to generate a nine-parameter fingerprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by describing novel functions to genes in mitosis and cytokinesis. In particular, we identified two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis in mammalian cells. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin. An esiRNA targeting LIN54 was transfected into HCT116 cells. RNA was harvested at 24, 48, and 72 hours post-transfection for microarray analysis. Transcripts regulated at least 1.5-fold, and with a p-value<0.01 were identified as signature transcripts.

2010-06-25 | E-GEOD-9176 | biostudies-arrayexpress

A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

Project description:Long non-coding RNAs (lncRNAs) regulate diverse biological pathways. Unlike protein coding genes, where methods to comprehensibly study their functional roles in cellular systems are available, techniques to systematically investigate lncRNAs have largely remained unexplored. Here, we report a technology for combined Knockdown and Localization Analysis of Non-coding RNAs (c-KLAN) that merges phenotypic characterization and localization approaches to study lncRNAs. Using a library of endoribonuclease prepared short interfering RNAs (esiRNAs) coupled with a pipeline for synthesizing labeled riboprobes for RNA fluorescence in situ hybridization (FISH), we demonstrate the utility of c-KLAN by identifying a novel transcript Panct1 (Pluripotency associated non-coding transcript 1) that regulates embryonic stem cell identity. We postulate that c-KLAN should be generally useful in the discovery of lncRNAs implicated in various biological processes. In this experiment, the levels of Panct1(AK156552) have been depleted by RNAi and the expression levels of all genes have been monitored 72 hours post transfection with esiRNAs against Panct1. An esiRNA against non-targeting Luciferase was used as a negative control.

2012-02-15 | E-GEOD-29798 | biostudies-arrayexpress

Genome-wide resources of endoribonucleotide-prepared short interfering RNAs for specific loss-of-function studies

Project description:RNA interference (RNAi) has become an important technique for loss-of-gene functionstudies in mammalian cells. To achieve reliable results in an RNAi experiment, efficientand specific silencing triggers are required. Here we present genome-wide data sets forthe production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. The database RiDDLE was installed for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective, and specific alternative to currently available mammalian RNAi resources. Consensus gene lists used to generate the clusters appearing in the associated publication have been linked as supplementary files at the foot of the Series record. There is a consensus genelist for each gene in the study: GSE6807_KIAA1387_ConsensusGeneList.txt GSE6807_MAPK14_ConsensusGeneList.txt Keywords: RNAi, off-target effects, esiRNA

2007-04-10 | GSE6807 | GEO

A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

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Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage stage chimaerism and mixolpoidy

Project description:Chimaerism and mixoploidy define the presence of cell lineages with different parental genomes or different ploidy states in a single individual. Our knowledge on their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation stage bovine embryos (n=23) following in vitro fertilization. Not only abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term “heterogoneic division” to indicate the events leading to non-canonical zygotic cytokinesis segregating the parental genomes into distinct lineages. Persistence of those cell lines during development is the likely cause of chimaerism and mixoploidy in mammals.

2016-03-11 | GSE76305 | GEO

Expression analysis of Candida albicans following a block in mitotic exit

Project description:Candida albicans demonstrates three main growth morphologies yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. We have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Consistent with its role in activating the mitotic exit network Tem1 localised to spindle pole bodies in a cell cycle dependent manner. Cells depleted of Tem1 displayed highly polarised growth but ultimately fail to complete cytokinesis and re-enter the cell cycle following nuclear division. At the transcriptional level genes downregulated following the depletion of Tem1 where significantly enriched for genes whose expression peaks early in the cell cycle and for those associated with glycolysis. For expression analysis triplicate RNA samples were generated from exponentially growing C. albicans yeast cells and from cells following 8 h inhibition of TEM1 expression. Gene expression was determined using RNA-seq on the Illumina HiSeq 2000 platform.

2014-01-01 | E-GEOD-48650 | biostudies-arrayexpress

Expression analysis of Candida albicans following a block in mitotic exit

2014-01-01 | GSE48650 | GEO

Chromatin immunoprecipitation of Bacillus subtilis to identify Noc protein binding sites

Project description:Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis the nucleoid occlusion protein Noc protects chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and show that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division but that its activity is strictly localized by interaction with NBS sites in vivo. We propose that Noc not only serves as a spatial regulator of cell division to protect the nucleoid, but also a timing device with an important role in the co-ordination of chromosome segregation and cell division.

2009-07-01 | E-MEXP-2133 | biostudies-arrayexpress

Cytokinetic Abscission in Toxoplasma gondii is governed by Protein Phosphatase 2A and the Daughter Cell Scaffold Complex

Project description:Cytokinetic abscission is the last step of cell division during which the daughter cells physically separate through the generation of barriers in the form of new plasma membrane or cell wall. While the contractile ring is a prominent structure during cytokinesis in bacteria, fungi and animal cells, the mechanism is divergent in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the intact mother cell by endodyogeny. The acquisition of plasma membrane at the end of the division cycle occurs by an unknown mechanism, when the mother cell disassembles, and the daughter cells emerge. Here we identified and functionally characterized the essential subunits of a predicted PP2A complex in T. gondii, named PP2A-2. The three subunits PP2A-A2, -B2 and -C2 exhibit a dynamic localization during parasite division. Their individual downregulation prevents the accumulation of plasma membrane at the division plane, resulting in a block of cytokinesis. Remarkably, the absence of cytokinetic abscission does not preclude division cycles and the consecutive progeny is able to successfully egress from the infected cells but fails to glide andinvade new host cells.

2024-06-13 | PXD051495 | Pride

Daughter Cell Scaffold proteins drive cytokinetic abscission in Toxoplasma gondii

Project description:Cytokinetic abscission is the last step of cell division during which the daughter cells physically separate through the generation of barriers in the form of new plasma membrane or cell wall. While the contractile ring is a prominent structure during cytokinesis in bacteria, fungi and animal cells, the mechanism is divergent in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the intact mother cell by endodyogeny. The acquisition of plasma membrane at the end of the division cycle occurs by an unknown mechanism, when the mother cell disassembles, and the daughter cells emerge. Here we identified and functionally characterized a complex of three essential T. gondii proteins, named Daughter Cell Scaffold proteins 1 and 2 (DCS1, DCS2) as well as PP2A-B2 (also named DCS3) that exhibit a dynamic localization during parasite division. Their individual downregulation prevents the accumulation of plasma membrane at the division plane, resulting in a block of cytokinesis. Remarkably, the absence of cytokinetic abscission does not preclude division cycles and the consecutive progeny is able to successfully egress from the infected cells but fails to glide and invade except for conjoined twin parasites.

2024-06-13 | PXD047907 | Pride

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