Genome-wide functional analysis of human cell-cycle regulators

Mukherji, Mridul; Bell, Russell; Supeková, Lubica; Wang, Yan; Orth, Anthony P.; Batalov, Serge; Miraglia, Loren; Huesken, Dieter; Lange, Joerg; Martin, Christopher; Sahasrabudhe, Sudhir; Reinhardt, Mischa; Natt, François; Hall, Jonathan; Mickanin, Craig; Labow, Mark; Chanda, Sumit K.; Cho, Charles Y.; Schultz, Peter G.

doi:10.1073/pnas.0604320103

Cited by 126 publications

(126 citation statements)

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“…5 In addition to single gene knockdown, investigators developed RNAi libraries to discover gene pathways controlling various cellular processes and facilitate global understanding of cellular behavior. 6 --9 This approach was used to identify genes that are involved in proteasome-mediated proteolysis, 10 virus infection and replication, 11 --14 cytokinesis, 8,9,15,16 endocytosis, 17 stem cell self-renewal 18 or cancer cell proliferation and survival. 19,20 RNAi knockdown requires a good selection strategy to link genetic information to cellular phenotype.…”

Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased aSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

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Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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“…Many 4 endogenous rice small RNAs were also found to be complementary to mouse (Mus musculus), 5 pig (Sus scrofa), chicken (Gallus gallus), and cow (Bos taurus) genes (Table 1). Table 3 lists 6 some genes that have been previously identified as potential regulators of the human cell cycle 7 through in vitro RNAi knockdown experiments (Mukherji et al, 2006;Kittler et al, 2007) and 8 that have perfect complementarity (or only one mismatch) to one or more small RNAs found in 9 rice grain. 10 11 Discussion: 12…”

Section: Results: 22mentioning

confidence: 99%

“…Among the human genes identified as having 3 high homology to small RNA molecules present in rice, several have been previously shown to 4 have an associated phenotype such as cell cycle arrest when suppressed in vitro using synthetic 5 siRNAs in cultured human cells (Mukherji et al, 2006;Kittler et al, 2007). In addition, rice 6 small RNAs had perfect complementarity to human genes encoding cell cycle regulators, 7 structural proteins and adhesion molecules, developmental regulators, growth factors, metabolic 8 enzymes/proteins, receptors, signal transduction proteins, transcription factors/transcriptional 9 regulators and transporters.…”

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Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Ivashuta¹,

Petrick²,

Heisel³

et al. 2009

Food and Chemical Toxicology

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23Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA 24 interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, 25 mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed 26 naturally and through conventional breeding to achieve desired plant phenotypes. The present 27 study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in 28 soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and 29 feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to 30 human genes as well as those of other mammals. The abundance of endogenous small RNA 31 molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice 32 ACCEPTED MANUSCRIPT 3 and the homology of a number of these dietary small RNAs to human and animal genomes and 1 transcriptomes establishes a history of safe consumption for dietary small RNAs. 2 3 Introduction: 4 RNA-mediated gene regulation (RNA interference, RNAi) is a highly conserved endogenous 5 mechanism for regulation of gene expression in eukaryotes that operates through multiple 6 pathways (Di Serio et al., 2001;Bantounas et al., 2004;Mello and Conte, 2004; Brodersen and 7 Voinnet, 2006;Mallory and Vaucheret, 2006). RNAi plays important roles in development, 8 pathogen defense and disease response in mammals, plants, and insects (Chang and Mendell, 9 2007;Pedersen et al., 2007). RNAi pathways are triggered by small RNAs that are usually 20 to 10 26 nucleotides (nt) long and are represented by diverse classes that differ from each other in their 11 biogenesis such as small interfering RNAs (siRNAs), microRNAs (miRNAs), trans-acting 12 siRNAs and other classes of small RNAs (Brodersen and Voinnet, 2006; Mallory and Vaucheret, 13 2006;Peters and Meister, 2007). The function of these various classes of small RNAs in animal 14 and plant RNAi pathways involves sequence-specific recruitment of the RNA silencing complex 15 to mRNA or DNA, leading to target mRNA cleavage, translational inhibition, or DNA 16 modifications ( Figure 1). Small RNA regulatory networks are highly conserved in plants and 17 animals and are an essential part of endogenous gene regulation. For example, it has been 18 predicted that endogenous miRNAs likely regulate expression of at least one third of all human 19 genes (Lewis et al., 2005). 20 21RNAi has been harnessed in the improvement of several conventional crops including soybean, 22 rice and maize. Soybean varieties that are precursors to those currently cultivated have a dark 23 ACCEPTED MANUSCRIPT 4 pigmentation due to anthocyanin content. Breeders have selected for soybeans with a yellow 1 seed coat attributed to RNAi-mediated suppression of the chalcone synthase gene (Tuteja et al., 2 2004). RNAi has also been attributed to a conventional low-glutelin (seed storage protein) rice 3 variety useful for those who must restrict dietary p...

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“…Genome-wide LOF screens are currently widely exploited biological tools, which generate a huge amount of data, but the analytical tools applied for processing these data and arriving at conclusions are generally not commensurate with the biological capability of this strong and expensive method. In practice, the results of such screens are either finally narrowed down to the discovery of 1-2 specific genes only, or, alternatively, undergo too-general systems biology analysis with conclusions barely (or not at all) meaningful with respect to real biological needs [5]- [14]. Here, we try to find a way between these Scylla and Charybdis, and to propose a powerful analytical tool, based on the concept of Specific Network, meaning a Network corresponding to a certain process and specific for a given cell system (e.g., given cell line, given cancer type, etc.).…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Growth Control Network Specific for Human Lung Adenocarcinoma Cells

Pinna

Zinovyev

Araujo

et al. 2012

Math. Model. Nat. Phenom.

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Abstract. Many cancer-associated genes and pathways remain to be identified in order to clarify the molecular mechanisms underlying cancer progression. In this area, genome-wide lossof-function screens appear to be powerful biological tools, allowing the accumulation of large amounts of data. However, this approach currently lacks analytical tools to exploit the data with maximum efficiency, for which systems biology methods analyzing complex cellular networks may be extremely helpful. In this article we report such a systems biology strategy based on the construction of a Network for a biological process and specific for a given cell system (cell type). The networks are created from genome-wide loss-of-function screen datasets. We also propose tools to analyze network properties. As one of the tools, we suggest a mathematical model for discrimination between two distinct cell processes that may be affected by knocking down the activity of a gene, i. e., a decreased cell number may be caused by arrested cell proliferation or enhanced cell death. Next we show how this discrimination between the two cell processes helps to construct two corresponding subnetworks. Finally, we demonstrate an application of the proposed strategy to the identification and characterization of putative novel genes and pathways significant for the control of lung cancer cell growth, based on the results of a genome-wide proliferation/viability loss-of-function screen of human lung adenocarcinoma cells.

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Genome-wide functional analysis of human cell-cycle regulators

Cited by 126 publications

References 36 publications

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Analysis of the Growth Control Network Specific for Human Lung Adenocarcinoma Cells

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