Quantitation of RNA by a fluorometric method using the SYTO RNASelect stain

Wu, Yexu; Liu, Yanfeng; Lu, Chuanchuan; Lei, Senlin; Li, Jianghua; Du, Guocheng

doi:10.1016/j.ab.2020.113857

Cited by 10 publications

(5 citation statements)

References 11 publications

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“…We next sought to evaluate whether the identification of RNAbinding proteins in tSV related to the release of tSV-containing RNA. We used RNASelect 52 , a probe fluorogenic upon binding to RNA, to selectively track the mobilization of RNA + structures to the synaptic pole and its release as part of tSV. We also used BODIPY-TR Ceramide to follow membrane structures associated with RNA.…”

Section: Resultsmentioning

confidence: 99%

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles

et al. 2022

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The immunological synapse is a molecular hub that facilitates the delivery of three activation signals, namely antigen, costimulation/corepression and cytokines, from antigen-presenting cells (APC) to T cells. T cells release a fourth class of signaling entities, trans-synaptic vesicles (tSV), to mediate bidirectional communication. Here we present bead-supported lipid bilayers (BSLB) as versatile synthetic APCs to capture, characterize and advance the understanding of tSV biogenesis. Specifically, the integration of juxtacrine signals, such as CD40 and antigen, results in the adaptive tailoring and release of tSV, which differ in size, yields and immune receptor cargo compared with steadily released extracellular vesicles (EVs). Focusing on CD40L+ tSV as model effectors, we show that PD-L1 trans-presentation together with TSG101, ADAM10 and CD81 are key in determining CD40L vesicular release. Lastly, we find greater RNA-binding protein and microRNA content in tSV compared with EVs, supporting the specialized role of tSV as intercellular messengers.

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Section: Resultsmentioning

confidence: 99%

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles

et al. 2022

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“…Hence, it could estimate the cellular RNA content of live yeast cells. 26 Here, we evaluated the relationship between fluorescence and cellular RNA content. Yeast cells with varying cellular RNA content were obtained by harvesting them at different growth stages.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…SYTO RNASelect is a cell membrane-permeant, RNA-specific dye. Hence, it could estimate the cellular RNA content of live yeast cells . Here, we evaluated the relationship between fluorescence and cellular RNA content.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Ribonucleic Acid Production by High-Throughput Screening Based on Fluorescence Activation and Transcriptomic-Guided Fermentation Optimization in Saccharomyces cerevisiae

Lei²,

et al. 2023

J. Agric. Food Chem.

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Currently, the primary source of ribonucleic acids (RNAs), which is used as a flavor enhancer and nutritional supplement in the food manufacturing and processing industries, for large-scale industrial production is yeast, where the challenge is to optimize the cellular RNA content. Here, we developed and screened yeast strains yielding abundant RNAs via various methods. The novel Saccharomyces cerevisiae strain H1 with a 45.1% higher cellular RNA content than its FX-2 parent was successfully generated. Comparative transcriptomic analysis elucidated the molecular mechanisms underlying RNA accumulation in H1. Upregulation of genes encoding the hexose monophosphate and sulfur-containing amino acid biosynthesis pathways promoted RNA accumulation in the yeast, particularly in the presence of glucose as the sole carbon source. Feeding methionine into the bioreactor resulted in 145.2 mg/g dry cell weight and 9.6 g/L of cellular RNA content, which is the highest volumetric productivity of RNAs achieved in S. cerevisiae. This strategy of breeding S. cerevisiae strain with a higher capacity of accumulating abundant RNAs did not employ any genetic modification and thus will be favored by the food industry.

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“…Since the models predicted a reduced mRNA pool in 1N cells, we performed live-cell staining with SYTO RNASelect (Wu et al, 2020), a fluorogenic RNA-specific dye that primarily (~70% of the signal) binds mRNAs in E. coli based on rifampicin treatment (Bakshi et al, 2014). In our RNASelect staining experiments, we mixed 1N cells with multi-N cells of similar size ranges prior to incubation with the dye to mitigate variability in staining.…”

Section: As Shown Inmentioning

confidence: 99%

Genome concentration limits cell growth and modulates proteome composition inEscherichia coli

Mäkelä,

Papagiannakis,

Lin

et al. 2024

Preprint

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Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication results in a decrease in the concentration of active RNA polymerases and ribosomes. The resulting sub-linear scaling of total active RNA polymerases and ribosomes with cell size leads to sub-exponential growth, even within physiological cell sizes. Cell growth rate scales proportionally with the total number of active ribosomes in a DNA concentration-dependent manner. Tandem-mass-tag mass spectrometry experiments further reveal that a decrease in DNA-to-cell-volume ratio proportionally remodels the composition of the proteome with cell size independently of the environment. Altogether, our findings indicate that genome concentration is an important driver of exponential cell growth and a global modulator of proteome composition in E. coli. Comparison with studies on eukaryotic cells suggests DNA concentration-dependent scaling principles of gene expression across domains of life.

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Quantitation of RNA by a fluorometric method using the SYTO RNASelect stain

Cited by 10 publications

References 11 publications

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles

Enhanced Ribonucleic Acid Production by High-Throughput Screening Based on Fluorescence Activation and Transcriptomic-Guided Fermentation Optimization in Saccharomyces cerevisiae

Genome concentration limits cell growth and modulates proteome composition inEscherichia coli

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