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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

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

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

2012-02-15 | GSE29798 | GEO

Defining how Pak1 regulates cell polarity and cell division in fission yeast

Project description:For polarized growth of cells, protein kinases regulate the assembly of cytoskeletal structures at the correct time and place. Large cytoskeletal structures must also be assembled in a regulated manner during cytokinesis and cell separation. We performed a visual screen to identify cell polarity protein kinases that might also function in cytokinesis and cell separation. We found that the Cdc42-activated protein kinase Pak1 (also called Orb2 and Shk1) colocalizes with the assembling cytokinetic ring and remains in the cell middle during septation. Mutations in pak1 led to defects in cytokinetic ring assembly and cell separation, as well as previously known cell polarity defects. We performed a phosphoproteomic screen and identified novel Pak1 targets that were verified as direct substrates in vitro. Top targets included proteins that function in polarized growth, cytokinesis, and septation. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the cell cortex and the cytokinetic ring. For cell separation, Pak1 phosphorylates the RNA-binding protein Sts5 to prevent its assembly into P-body granules. The cell separation defect of pak1 mutants was suppressed by a non-phosphorylatable sts5 mutant. These results show that Pak1 acts directly on components of the cytokinetic ring, but unexpectedly promotes the later stages of cell division by inhibiting the assembly of ribonucleoprotein granules. More broadly, our work reveals that cell polarity signaling proteins coordinate diverse events to promote cell division at the end of the cell cycle.

2019-07-02 | MSV000084047 | MassIVE

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

Single-cell RNA-sequencing of wildtype murine tracheal epithelial cells cultured at air-liquid interace for one, three, nine, and thirty-six days.

Project description:The canonical mitotic cell cycle coordinates cell growth, DNA replication, centriole duplication and cytokinesis to generate two cells from one. In certain contexts, such as in mammalian trophoblast giant cells or hepatocytes, cells employ cell cycle variants like the endocycle or endomitosis to increase DNA content without undergoing cytokinesis. Multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tracts, generate hundreds of centrioles during differentiation, each of which extend a motile cilium. We found that multiciliated cells utilize a variant of the cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys much of the mitotic cell cycle regulatory framework, including cell division kinases (CDKs) and cyclins, to generate hundreds of centrioles without undergoing DNA synthesis or cytokinesis. Unlike the mitotic cycle, a transcriptional repressor, E2F7, is upregulated during the multiciliation cycle. E2F7 directly represses genes encoding DNA synthesis machinery during the multiciliation cycle. Loss of E2F7 results in aberrant DNA synthesis and disruption of centriole biogenesis in differentiating multiciliated cells. Thus, multiciliation is coordinated by a variant cell cycle that uses E2F7 to uncouple centriole synthesis from DNA replication.

2024-03-30 | GSE228109 | GEO

E2F7-GFP binding sites in primary airway mouse epithelial cells (mTECs) cultured at air-liquid interface for three days.

2024-03-30 | GSE245742 | GEO

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