Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.

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Dual functions of Rack1 in regulating Hedgehog pathway

Cell Death and Differentiation ◽

10.1038/s41418-020-0563-7 ◽

2020 ◽

Vol 27 (11) ◽

pp. 3082-3096 ◽

Cited By ~ 1

Author(s):

Yan Li ◽

Xiaohan Sun ◽

Dongqing Gao ◽

Yan Ding ◽

Jinxiao Liu ◽

...

Keyword(s):

Mammalian Cells ◽

Cellular Localization ◽

Multiple Roles ◽

Dual Roles ◽

Cubitus Interruptus ◽

Trimeric Complex ◽

Physiological Processes ◽

Underlying Mechanisms ◽

The Stability ◽

Fine Tune

Abstract Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

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The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke

Biochemical Journal ◽

10.1042/bj1590585 ◽

1976 ◽

Vol 159 (3) ◽

pp. 585-600 ◽

Cited By ~ 4

Author(s):

K S Chapman ◽

J Ingle

Keyword(s):

Mammalian Cells ◽

Molecular Size ◽

Rrna Synthesis ◽

Kinetic Measurements ◽

Polyadenylic Acid ◽

Nuclear Rna ◽

The Stability ◽

Time Required ◽

Total Tissue ◽

Isopycnic Centrifugation

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 × 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 × 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 × 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 × 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.

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Kinetochore-microtubule stability governs the metaphase requirement for Eg5

Molecular Biology of the Cell ◽

10.1091/mbc.e14-03-0785 ◽

2014 ◽

Vol 25 (13) ◽

pp. 2051-2060 ◽

Cited By ~ 26

Author(s):

A. Sophia Gayek ◽

Ryoma Ohi

Keyword(s):

Physical Properties ◽

Human Cell ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Human Cell Lines ◽

Bipolar Spindle ◽

Daughter Cells ◽

Microtubule Stability ◽

The Stability ◽

Bipolar Spindles

The mitotic spindle is a bipolar, microtubule (MT)-based cellular machine that segregates the duplicated genome into two daughter cells. The kinesin-5 Eg5 establishes the bipolar geometry of the mitotic spindle, but previous work in mammalian cells suggested that this motor is unimportant for the maintenance of spindle bipolarity. Although it is known that Kif15, a second mitotic kinesin, enforces spindle bipolarity in the absence of Eg5, how Kif15 functions in this capacity and/or whether other biochemical or physical properties of the spindle promote its bipolarity have been poorly studied. Here we report that not all human cell lines can efficiently maintain bipolarity without Eg5, despite their expressing Kif15. We show that the stability of chromosome-attached kinetochore-MTs (K-MTs) is important for bipolar spindle maintenance without Eg5. Cells that efficiently maintain bipolar spindles without Eg5 have more stable K-MTs than those that collapse without Eg5. Consistent with this observation, artificial destabilization of K-MTs promotes spindle collapse without Eg5, whereas stabilizing K-MTs improves bipolar spindle maintenance without Eg5. Our findings suggest that either rapid K-MT turnover pulls poles inward or slow K-MT turnover allows for greater resistance to inward-directed forces.

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Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells

Angewandte Chemie International Edition ◽

10.1002/anie.201712284 ◽

2018 ◽

Vol 57 (8) ◽

pp. 2165-2169 ◽

Cited By ~ 85

Author(s):

Simon Dzatko ◽

Michaela Krafcikova ◽

Robert Hänsel-Hertsch ◽

Tomas Fessl ◽

Radovan Fiala ◽

...

Keyword(s):

Mammalian Cells ◽

The Stability

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Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells

Molecular Biology of the Cell ◽

10.1091/mbc.e14-11-1521 ◽

2015 ◽

Vol 26 (6) ◽

pp. 1072-1083 ◽

Cited By ~ 21

Author(s):

Souvik Ghosh ◽

Mainak Bose ◽

Anirban Ray ◽

Suvendra N. Bhattacharyya

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Cell Growth ◽

Subcellular Distribution ◽

Mammalian Cells ◽

Cellular Response ◽

Mature Mirnas ◽

Regulatory Rnas ◽

The Stability ◽

External Stimuli

MicroRNAs (miRNAs) are tiny posttranscriptional regulators of gene expression in metazoan cells, where activity and abundance of miRNAs are tightly controlled. Regulated turnover of these regulatory RNAs is important to optimize cellular response to external stimuli. We report that the stability of mature miRNAs increases inversely with cell proliferation, and the increased number of microribonucleoproteins (miRNPs) in growth-restricted mammalian cells are in turn associated with polysomes. This heightened association of miRNA with polysomes also elicits reduced degradation of target mRNAs and impaired extracellular export of miRNA via exosomes. Overall polysome sequestration contributes to an increase of cellular miRNA levels but without an increase in miRNA activity. Therefore miRNA activity and turnover can be controlled by subcellular distribution of miRNPs that may get differentially regulated as a function of cell growth in mammalian cells.

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The NR1 subunit of the N-methyl-d-aspartate receptor can be efficiently expressed alone in the cell surface of mammalian cells and is required for the transport of the NR2A subunit

Biochemical Journal ◽

10.1042/bj3560539 ◽

2001 ◽

Vol 356 (2) ◽

pp. 539-547 ◽

Cited By ~ 5

Author(s):

Mónica GARCÍA-GALLO ◽

Jaime RENART ◽

Margarita DÍAZ-GUERRA

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Half Life ◽

Mammalian Cells ◽

Maturation Process ◽

Aspartate Receptor ◽

Heterologous System ◽

The Stability ◽

Nr2a Subunit ◽

In Cells

We have used a heterologous system of expression of N-methyl-d-aspartate (NMDA) receptors based on the use of vaccinia virus to analyse the maturation, transport, assembly and differential expression of the NR1 and NR2A subunits of the receptors. We have demonstrated that the NR1 subunit is efficiently transported to the plasma membrane in cells expressing NR1 alone, similarly to cells producing NR1 and NR2A together. In contrast, NR2A requires NR1 expression to be located at the cell surface. The stability of both receptor subunits expressed alone is similar to that obtained in cells producing NR1 and NR2A. In pulse–chase experiments, the NR1 subunit displays a biphasic decay, with a fraction of the protein having a half-life of only 1h and the remaining presenting a turnover longer than 24h, similar to values obtained for the NR2A subunit. Our results also show a maturation process affecting the carbohydrate moiety in the NR1 subunit, such that immature NR1has a much shorter half-life than the mature form or the NR2A subunit. Finally, we show that only a fraction of mature NR1 interacts with NR2A to form multimeric functional complexes.

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NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

10.1101/358911 ◽

2018 ◽

Author(s):

Yuehong Yang ◽

Wei Wang ◽

Min Li ◽

Wen Zhang ◽

Yuliang Huang ◽

...

Keyword(s):

Atpase Activity ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Mammalian Cells ◽

Ectopic Expression ◽

Sister Chromatid Cohesion ◽

Chaperone Function ◽

Chromatid Cohesion ◽

Protein Instability ◽

The Stability

AbstractSister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

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Low Expression of YTH Domain-Containing 1 Promotes Microglial M1 Polarization by Reducing the Stability of Sirtuin 1 mRNA

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.774305 ◽

2021 ◽

Vol 15 ◽

Author(s):

Hongxiu Zhou ◽

Zongren Xu ◽

Xingyun Liao ◽

Shiyun Tang ◽

Na Li ◽

...

Keyword(s):

Inflammatory Response ◽

Nuclear Export ◽

Mammalian Cells ◽

Sirtuin 1 ◽

Sequencing Data ◽

Microglial Polarization ◽

M1 Polarization ◽

Stat3 Phosphorylation ◽

Yth Domain ◽

The Stability

The N6-methyladenosine (m6A) modification is the most abundant posttranscriptional mRNA modification in mammalian cells and is dynamically modulated by a series of “writers,” “erasers,” and “readers.” Studies have shown that m6A affects RNA metabolism in terms of RNA processing, nuclear export, translation, and decay. However, the role of the m6A modification in retinal microglial activation remains unclear. Here, we analyzed the single-cell RNA sequencing data of retinal cells from mice with uveitis and found that the m6A-binding protein YTH domain-containing 1 (YTHDC1) was significantly downregulated in retinal microglia in the context of uveitis. Further studies showed that YTHDC1 deficiency resulted in M1 microglial polarization, an increased inflammatory response and the promotion of microglial migration. Mechanistically, YTHDC1 maintained sirtuin 1 (SIRT1) mRNA stability, which reduced signal transducer and activator of transcription 3 (STAT3) phosphorylation, thus inhibiting microglial M1 polarization. Collectively, our data show that YTHDC1 is critical for microglial inflammatory response regulation and can serve as a target for the development of therapeutics for autogenic immune diseases.

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Polyfluorene-Based Multicolor Fluorescent Nanoparticles Activated by Temperature for Bioimaging and Drug Delivery

Nanomaterials ◽

10.3390/nano9101485 ◽

2019 ◽

Vol 9 (10) ◽

pp. 1485 ◽

Cited By ~ 2

Author(s):

Marta Rubio-Camacho ◽

Yolanda Alacid ◽

Ricardo Mallavia ◽

María José Martínez-Tomé ◽

C. Reyes Mateo

Keyword(s):

Mammalian Cells ◽

Photophysical Properties ◽

Drug Carriers ◽

Fluorescent Nanoparticles ◽

Tem Analysis ◽

Transmission Electron ◽

Hydrophilic Drug ◽

Thermosensitive Liposomes ◽

Size And Morphology ◽

The Stability

Multifunctional nanoparticles have been attracting growing attention in recent years because of their capability to integrate materials with different features in one entity, which leads them to be considered as the next generation of nanomedicine. In this work, we have taken advantage of the interesting properties of conjugated polyelectrolytes to develop multicolor fluorescent nanoparticles with integrating imaging and therapeutic functionalities. With this end, thermosensitive liposomes were coated with three recently synthesized polyfluorenes: copoly-((9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(phenylene)) bromide (HTMA-PFP), copoly-((9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-2,7-(fluorene)-alt-4,7-(2- (phenyl)benzo(d) (1,2,3) triazole)) bromide (HTMA-PFBT) and copoly-((9,9-bis(6′-N,N,N- trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(naphtho(2,3c)-1,2,5-thiadiazole)) bromide (HTMA-PFNT), in order to obtain blue, green and red fluorescent drug carriers, respectively. The stability, size and morphology of the nanoparticles, as well as their thermotropic behavior and photophysical properties, have been characterized by Dynamic Light Scattering (DLS), Zeta Potential, transmission electron microscope (TEM) analysis and fluorescence spectroscopy. In addition, the suitability of the nanostructures to carry and release their contents when triggered by hyperthermia has been explored by using carboxyfluorescein as a hydrophilic drug model. Finally, preliminary experiments with mammalian cells demonstrate the capability of the nanoparticles to mark and visualize cells with different colors, evidencing their potential use for imaging and therapeutic applications.

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