scholarly journals Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors

2020 ◽  
Vol 295 (44) ◽  
pp. 15112-15133
Author(s):  
Elizabeth T. Abshire ◽  
Kelsey L. Hughes ◽  
Rucheng Diao ◽  
Sarah Pearce ◽  
Shreekara Gopalakrishna ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Tissue Type ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Mitochondrial Functions ◽  
A Cell ◽  
Necessary And Sufficient ◽  
The Impact ◽  
Expression Processing ◽  
In Cells

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type–specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells.

scholarly journals Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites

2020 ◽  
Author(s):  
Elizabeth T. Abshire ◽  
Kelsey Hughes ◽  
Rucheng Diao ◽  
Sarah Pearce ◽  
Raymond C. Trievel ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Tissue Type ◽  
Mitochondrial Localization ◽  
Cellular Functions ◽  
Differential Processing ◽  
Specific Manner ◽  
A Cell ◽  
Necessary And Sufficient ◽  
The Impact ◽  
Expression Processing

ABSTRACTNocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We demonstrate that the NOCT protein is differentially expressed and processed in a cell and tissue type specific manner as a means to control its localization to the cytoplasm or mitochondria. Our studies also show that the N-terminus of NOCT is necessary and sufficient to confer mitochondrial localization. We then measured the impact of cytoplasmic NOCT on the transcriptome and report that it regulates the levels of hundreds of mRNAs that are enriched for components of signaling pathways, neurological functions, and regulators of osteoblast differentiation. Recent biochemical data indicate that NOCT dephosphorylates nicotinamide adenine dinucleotide (NAD) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in manner specified by its intracellular localization.

scholarly journals The kinetic landscape of an RNA binding protein in cells

2020 ◽  
Author(s):  
Deepak Sharma ◽  
Leah L. Zagore ◽  
Matthew M. Brister ◽  
Xuan Ye ◽  
Carlos E. Crespo-Hernández ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cumulative Probability ◽  
Mrna Levels ◽  
Dissociation Kinetics ◽  
The Impact ◽  
In Cells

ABSTRACTGene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions 2. However, these kinetics were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how previously inaccessible, kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.

scholarly journals Effects of Pesticides on Longevity and Bioenergetics in Invertebrates—The Impact of Polyphenolic Metabolites

2021 ◽  
Vol 22 (24) ◽  
pp. 13478
Author(s):  
Fabian Schmitt ◽  
Lukas Babylon ◽  
Fabian Dieter ◽  
Gunter P. Eckert
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Stress Resistance ◽  
Expression Patterns ◽  
Hazardous Substances ◽  
Mrna Levels ◽  
Atp Levels ◽  
Mitochondrial Functions ◽  
Heat Stress Resistance ◽  
The Impact

Environmentally hazardous substances such as pesticides are gaining increasing interest in agricultural and nutritional research. This study aims to investigate the impact of these compounds on the healthspan and mitochondrial functions in an invertebrate in vivo model and in vitro in SH-SY5Y neuroblastoma cells, and to investigate the potential of polyphenolic metabolites to compensate for potential impacts. Wild-type nematodes (Caenorhabditis elegans, N2) were treated with pesticides such as pyraclostrobin (Pyr), glyphosate (Gly), or fluopyram (Fluo). The lifespans of the nematodes under heat stress conditions (37 °C) were determined, and the chemotaxis was assayed. Energetic metabolites, including adenosine triphosphate (ATP), lactate, and pyruvate, were analyzed in lysates of nematodes and cells. Genetic expression patterns of several genes associated with lifespan determination and mitochondrial parameters were assessed via qRT-PCR. After incubation with environmentally hazardous substances, nematodes were incubated with a pre-fermented polyphenol mixture (Rechtsregulat®Bio, RR) or protocatechuic acid (PCA) to determine heat stress resistance. Treatment with Pyr, Glyph and Fluo leads to dose-dependently decreased heat stress resistance, which was significantly improved by RR and PCA. The chemotaxes of the nematodes were not affected by pesticides. ATP levels were not significantly altered by the pesticides, except for Pyr, which increased ATP levels after 48 h leads. The gene expression of healthspan and mitochondria-associated genes were diversely affected by the pesticides, while Pyr led to an overall decrease of mRNA levels. Over time, the treatment of nematodes leads to a recovery of the nematodes on the mitochondrial level but not on stress resistance on gene expression. Fermented extracts of fruits and vegetables and phenolic metabolites such as PCA seem to have the potential to recover the vitality of C. elegans after damage caused by pesticides.

scholarly journals The Impact of Elastic Deformations of the Extracellular Matrix on Cell Migration

2020 ◽  
Vol 82 (4) ◽  
Author(s):  
A. A. Malik ◽  
B. Wennberg ◽  
P. Gerlee
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Human Cancer ◽  
Matrix Stiffness ◽  
Human Cancer Cells ◽  
Adhesion Sites ◽  
The Matrix ◽  
A Cell ◽  
Necessary And Sufficient ◽  
The Impact

Abstract The mechanical properties of the extracellular matrix, in particular its stiffness, are known to impact cell migration. In this paper, we develop a mathematical model of a single cell migrating on an elastic matrix, which accounts for the deformation of the matrix induced by forces exerted by the cell, and investigate how the stiffness impacts the direction and speed of migration. We model a cell in 1D as a nucleus connected to a number of adhesion sites through elastic springs. The cell migrates by randomly updating the position of its adhesion sites. We start by investigating the case where the cell springs are constant, and then go on to assuming that they depend on the matrix stiffness, on matrices of both uniform stiffness as well as those with a stiffness gradient. We find that the assumption that cell springs depend on the substrate stiffness is necessary and sufficient for an efficient durotactic response. We compare simulations to recent experimental observations of human cancer cells exhibiting durotaxis, which show good qualitative agreement.

scholarly journals Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions

2015 ◽  
Vol 21 (3) ◽  
pp. 269-276 ◽  
Author(s):  
C. Agarinis ◽  
V. Orsini ◽  
P. Megel ◽  
Y. Abraham ◽  
H. Yang ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Hippo Pathway ◽  
Nuclear Pore ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Putative Gene ◽  
Nuclear Shuttling ◽  
The Hippo Pathway ◽  
Upstream Regulators ◽  
Transcription Coactivator

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic–nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.

scholarly journals Using Polysome Isolation with Mechanism Alteration to Uncover Transcriptional and Translational Dynamics in Key Genes

2014 ◽  
Author(s):  
Bradly Alicea
Keyword(s):  
Experimental Treatment ◽  
Process Variable ◽  
Human Cells ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Mrna Extraction ◽  
Drug Treatments ◽  
A Cell ◽  
Cell Type Specific

What does it mean when we say a cell?s biochemistry is regulated during changes to the phenotype? While there are a plethora of potential mechanisms and contributions to the final outcome, a more tractable approach is to examine the dynamics of mRNA. This way, we can assess the contributions of both known and unknown decay and aggregation processes for maintaining levels of gene product on a gene-by-gene basis. In this extended protocol, drug treatments that target specific cellular functions (termed mechanism disruption) can be used in tandem with mRNA extraction from the polysome to look at the dynamics of mRNA levels associated with transcription and translation at multiple stages during a physiological perturbation. This is accomplished through validating the polysome isolation method in human cells and comparing fractions of mRNA for each experimental treatment at multiple points in time. First, three different drug treatments corresponding to the arrest of various cellular processes are administered to populations of human cells. For each treatment, the transcriptome and translatome are compared directly at different time points by assaying both cell-type specific and non-specific genes. There are two findings of note. First, extraction of mRNA from the polysome and comparison with the transcriptome can yield interesting information about the regulation of cellular mRNA during a functional challenge to the cell. In addition, the conventional application of such drugs to assess mRNA decay is an incomplete picture of how severely challenged or senescent cells regulate mRNA in response. This extended protocol demonstrates how the gene- and process-variable degradation of mRNA might ultimately require investigations into the course-grained dynamics of cellular mRNA, from transcription to ribosome.

scholarly journals Cannabinoid-Induced Immunomodulation during Viral Infections: A Focus on Mitochondria

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 875
Author(s):  
Cherifa Beji ◽  
Hamza Loucif ◽  
Roman Telittchenko ◽  
David Olagnier ◽  
Xavier Dagenais-Lussier ◽  
...  
Keyword(s):  
Immune System ◽  
Viral Infections ◽  
Cell Types ◽  
Cellular Functions ◽  
Neuroprotective Effects ◽  
Potential Implication ◽  
Mitochondrial Functions ◽  
Negative Impacts ◽  
The Impact ◽  
Hiv 1

This review examines the impact of cannabinoids on viral infections, as well as its effects on the mitochondria of the nervous and immune system. The paper conveys information about the beneficial and negative impacts of cannabinoids on viral infections, especially HIV-1. These include effects on the inflammatory response as well as neuroprotective effects. We also explore non-apoptotic mitochondrial pathways modulated by the activity of cannabinoids, resulting in modifications to cellular functions. As a large part of the literature derives from studies of the nervous system, we first compile the information related to mitochondrial functions in this system, particularly through the CB1 receptor. Finally, we reflect on how this knowledge could complement what has been demonstrated in the immune system, especially in the context of the CB2 receptor and Ca2+ uptake. The overall conclusion of the review is that cannabinoids have the potential to affect a broad range of cell types through mitochondrial modulation, be it through receptor-specific action or not, and that this pathway has a potential implication in cases of viral infection.

scholarly journals Impressic Acid Ameliorates Atopic Dermatitis-Like Skin Lesions by Inhibiting ERK1/2-Mediated Phosphorylation of NF-κB and STAT1

2021 ◽  
Vol 22 (5) ◽  
pp. 2334
Author(s):  
Jae Ho Choi ◽  
Gi Ho Lee ◽  
Sun Woo Jin ◽  
Ji Yeon Kim ◽  
Yong Pil Hwang ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Skin Lesions ◽  
Histopathological Analysis ◽  
Mrna Levels ◽  
Chemokine Production ◽  
Dermal Thickness ◽  
Skin Symptoms ◽  
Tnf Α ◽  
Ifn Γ ◽  
In Cells

Impressic acid (IPA), a lupane-type triterpenoid from Acanthopanax koreanum, has many pharmacological activities, including the attenuation of vascular endothelium dysfunction, cartilage destruction, and inflammatory diseases, but its influence on atopic dermatitis (AD)-like skin lesions is unknown. Therefore, we investigated the suppressive effect of IPA on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin symptoms in mice and the underlying mechanisms in cells. IPA attenuated the DNCB-induced increase in the serum concentrations of IgE and thymic stromal lymphopoietin (TSLP), and in the mRNA levels of thymus and activation regulated chemokine(TARC), macrophage derived chemokine (MDC), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in mice. Histopathological analysis showed that IPA reduced the epidermal/dermal thickness and inflammatory and mast cell infiltration of ear tissue. In addition, IPA attenuated the phosphorylation of NF-κB and IκBα, and the degradation of IκBα in ear lesions. Furthermore, IPA treatment suppressed TNF-α/IFN-γ-induced TARC expression by inhibiting the NF-κB activation in cells. Phosphorylation of extracellular signalregulated protein kinase (ERK1/2) and the signal transducer and activator of transcription 1 (STAT1), the upstream signaling proteins, was reduced by IPA treatment in HaCaT cells. In conclusion, IPA ameliorated AD-like skin symptoms by regulating cytokine and chemokine production and so has therapeutic potential for AD-like skin lesions.

Sign in / Sign up

Export Citation Format

Share Document