scholarly journals QMAP-Seq: Quantitative and Multiplexed Analysis of Phenotype by Sequencing

2020 ◽  
Author(s):  
Sonia Brockway ◽  
Geng Wang ◽  
Jasen M. Jackson ◽  
David R. Amici ◽  
Seesha R. Takagishi ◽  
...  
Keyword(s):  
Stress Response ◽  
Mammalian Cells ◽  
Genetic Interaction ◽  
Cellular Stress Response ◽  
Model Organisms ◽  
Response Factors ◽  
Genetic Profiling ◽  
Chemical Genetic ◽  
Functional Relationships ◽  
Multiplexed Analysis

Abstract Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types—comprising 12 genetic perturbations in five cell lines—with 1,440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.

scholarly journals Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonia Brockway ◽  
Geng Wang ◽  
Jasen M. Jackson ◽  
David R. Amici ◽  
Seesha R. Takagishi ◽  
...  
Keyword(s):  
Stress Response ◽  
Mammalian Cells ◽  
Genetic Interaction ◽  
Cell Types ◽  
Cellular Stress Response ◽  
Model Organisms ◽  
Response Factors ◽  
Genetic Profiling ◽  
Chemical Genetic ◽  
Functional Relationships

AbstractChemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types—comprising 12 genetic perturbations in five cell lines—with 1440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.

scholarly journals m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Vol 22 (6) ◽  
pp. 2941
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

scholarly journals Vimentin protects differentiating stem cells from stress

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sundararaghavan Pattabiraman ◽  
Gajendra Kumar Azad ◽  
Triana Amen ◽  
Shlomi Brielle ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stress Response ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Cellular Stress Response ◽  
Cell Stiffness ◽  
Asymmetric Partitioning ◽  
Organismal Development

Abstract Vimentin is one of the first cytoplasmic intermediate filaments to be expressed in mammalian cells during embryogenesis, but its role in cellular fitness has long been a mystery. Vimentin is acknowledged to play a role in cell stiffness, cell motility, and cytoplasmic organization, yet it is widely considered to be dispensable for cellular function and organismal development. Here, we show that Vimentin plays a role in cellular stress response in differentiating cells, by recruiting aggregates, stress granules, and RNA-binding proteins, directing their elimination and asymmetric partitioning. In the absence of Vimentin, pluripotent embryonic stem cells fail to differentiate properly, with a pronounced deficiency in neuronal differentiation. Our results uncover a novel function for Vimentin, with important implications for development, tissue homeostasis, and in particular, stress response.

scholarly journals Flavonoids as Putative Inducers of the Transcription Factors Nrf2, FoxO, and PPARγ

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Kathrin Pallauf ◽  
Nils Duckstein ◽  
Mario Hasler ◽  
Lars-Oliver Klotz ◽  
Gerald Rimbach
Keyword(s):  
Transcription Factors ◽  
Free Radical ◽  
Stress Response ◽  
Cellular Response ◽  
Radical Scavenging ◽  
Free Radical Scavenging ◽  
Cellular Stress Response ◽  
Model Organisms ◽  
Luciferase Reporter ◽  
Hek 293

Dietary flavonoids have been shown to extend the lifespan of some model organisms and may delay the onset of chronic ageing-related diseases. Mechanistically, the effects could be explained by the compounds scavenging free radicals or modulating signalling pathways. Transcription factors Nrf2, FoxO, and PPARγpossibly affect ageing by regulating stress response, adipogenesis, and insulin sensitivity. Using Hek-293 cells transfected with luciferase reporter constructs, we tested the potency of flavonoids from different subclasses (flavonols, flavones, flavanols, and isoflavones) to activate these transcription factors. Under cell-free conditions (ABTS and FRAP assays), we tested their free radical scavenging activities and usedα-tocopherol and ascorbic acid as positive controls. Most of the tested flavonoids, but not the antioxidant vitamins, stimulated Nrf2-, FoxO-, and PPARγ-dependent promoter activities. Flavonoids activating Nrf2 also tended to induce a FoxO and PPARγresponse. Interestingly, activation patterns of cellular stress response by flavonoids were not mirrored by their activities in ABTS and FRAP assays, which depended mostly on hydroxylation in the flavonoid B ring and, in some cases, extended that of the vitamins. In conclusion, the free radical scavenging properties of flavonoids do not predict whether these molecules can stimulate a cellular response linked to activation of longevity-associated transcription factors.

scholarly journals Genetic interaction of the stress response factors ChAP1 and Skn7 in the maize pathogenCochliobolus heterostrophus

2013 ◽  
Vol 350 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Samer Shalaby ◽  
Olga Larkov ◽  
Netta Li Lamdan ◽  
Benjamin A. Horwitz
Keyword(s):  
Stress Response ◽  
Genetic Interaction ◽  
Response Factors

scholarly journals Coessential Genetic Networks Reveal the Organization and Constituents of a Dynamic Cellular Stress Response

2019 ◽  
Author(s):  
David R. Amici ◽  
Jasen M. Jackson ◽  
Kyle A. Metz ◽  
Daniel J. Ansel ◽  
Roger S. Smith ◽  
...  
Keyword(s):  
Stress Response ◽  
Biological Networks ◽  
Genotoxic Stress ◽  
Cellular Stress Response ◽  
Genetic Screens ◽  
Master Regulators ◽  
Functional Relationships ◽  
Response Network ◽  
The Face ◽  
Genome Scale

SummaryThe interrelated programs essential for cellular fitness in the face of stress are critical to understanding tumorigenesis, neurodegeneration, and aging. However, modelling the combinatorial landscape of stresses experienced by diseased cells is challenging, leaving functional relationships within the global stress response network incompletely understood. Here, we leverage genome-scale fitness screening data from 625 cancer cell lines, each representing a unique biological context, to build a network of “coessential” gene relationships centered around master regulators of the response to proteotoxic, oxidative, hypoxic, and genotoxic stress. This approach organizes the stress response into functional modules, identifies genes connecting distinct modules, and reveals mechanisms underlying cellular dependence on individual modules. As an example of the power of this approach, we discover that the previously unannotated HAPSTR (C16orf72) promotes resilience to diverse stressors as a stress-inducible regulator of the E3 ligase HUWE1. Altogether, we present a broadly applicable framework and interactive tool (http://fireworks.mendillolab.org/) to interrogate biological networks using unbiased genetic screens.

scholarly journals Cellular roles of the human Obg-like ATPase 1 (hOLA1) and its YchF homologs

2020 ◽  
Vol 98 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Nirujah Balasingam ◽  
Harland E. Brandon ◽  
Joseph A. Ross ◽  
Hans-Joachim Wieden ◽  
Nehal Thakor
Keyword(s):  
Stress Response ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Mrna Translation ◽  
Nucleotide Binding ◽  
Structure And Function ◽  
Cellular Stress Response ◽  
Related Family ◽  
And Function ◽  
Nucleotide Binding Proteins

P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis.

scholarly journals m5U54 tRNA Hypomodification by lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification, resulting in angiogenin (ANG) dependent tsRNA formation. More specifically, m5U54 hypomodification is followed by ANG overexpression and tRNA cleavage near the anticodon, resulting in accumulation of 5’tRNA-derived stress-induced RNAs (5’tiRNAs), in particular 5’tiRNA-GlyGCC and 5’tiRNA-GluCTC. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tsRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tsRNA formation in mammalian cells. These results establish a link between tRNA demethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Apoptosis and development

2003 ◽  
Vol 39 ◽  
pp. 11-24 ◽  
Author(s):  
Justin V McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cell Number ◽  
Model Organisms ◽  
Biological Processes ◽  
Nematode Caenorhabditis Elegans ◽  
Apoptotic Pathway ◽  
Genetic Pathways ◽  
Evolutionarily Conserved ◽  
Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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