scholarly journals Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling

2021 ◽  
Author(s):  
Paulo A Gameiro ◽  
Vesela Encheva ◽  
Mariana Silva dos Santos ◽  
James I MacRae ◽  
Jernej Ule
Keyword(s):  
Small Rnas ◽  
Mammalian Cells ◽  
Tandem Mass ◽  
Dynamic Features ◽  
13C Labeling ◽  
Stationary Conditions ◽  
Base Modifications ◽  
Labeling Approach ◽  
Kinetics Of ◽  
Combined Measurements

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. Yet, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications poorly understood. We developed a 13C labeling approach, 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleosides, and showed the distinct kinetics of N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits a distinct turnover in small RNAs and free ribonucleosides when compared to the known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of origin of modified RNAs at steady-state and their dynamics under non-stationary conditions.

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 Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry

2008 ◽  
Vol 49 (5) ◽  
pp. 1113-1125 ◽  
Author(s):  
Christopher A. Haynes ◽  
Jeremy C. Allegood ◽  
Kacee Sims ◽  
Elaine W. Wang ◽  
M. Cameron Sullards ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Electrospray Ionization ◽  
Mammalian Cells ◽  
Fatty Acyl ◽  
Tandem Mass ◽  
Ionization Tandem Mass Spectrometry

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Washout Kinetics of Viral Vectors from Cultured Mammalian Cells

2012 ◽  
Vol 17 (4) ◽  
pp. 188-197 ◽  
Author(s):  
Claudia Bagutti ◽  
Martin Schmidlin ◽  
Matthias Mueller ◽  
Peter Brodmann
Keyword(s):  
Mammalian Cells ◽  
Viral Vectors ◽  
Kinetics Of

The Leeuwenhoek Lecture, 1978 Bacteria as proper subjects for cancer research

1980 ◽  
Vol 208 (1171) ◽  
pp. 121-133 ◽  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Research ◽  
Mammalian Cells ◽  
Cell Renewal ◽  
Epigenetic Alterations ◽  
Cell Lineages ◽  
Abnormal Cells ◽  
Kinetics Of

Cancers are clones of abnormal cells, arising presumably as the result of mutational or epigenetic alterations of gene expression. The kinetics of appearance of spontaneous cancers in populations of multiplying cells (i. e. the relation between age and cancer incidence) will therefore depend, among other things, on how these populations are organized and, in general, on the kinetics of the response of cells to prolonged mutagenesis. The organization of cell renewal in epithelia (i. e. the arrangement of cell lineages) is still rather obscure; in particular, it is not known to what extent the properties and organization of the stem cells tend to protect them from accumulating mutations. We have tried to mimic the arrangement of epithelia by attaching multiplying bacteria to filters. Study of mutagenesis in long-term cultures of such anchored bacteria has led to the discovery of some additional pathways for DNA repair which also appear to operate in mammalian cells.

The kinetics of the B cyclin p56cdc13 and the phosphatase p80cdc25 during the cell cycle of the fission yeast Schizosaccharomyces pombe

1996 ◽  
Vol 109 (6) ◽  
pp. 1647-1653 ◽  
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Size Control ◽  
Base Line ◽  
Wild Type ◽  
Main Band ◽  
The Times ◽  
G1 Cells ◽  
Mutant Cells ◽  
Kinetics Of

The levels of the B cyclin p56cdc13 and the phosphatase p80cdc25 have been followed in selection-synchronised cultures of Schizosaccharomyces pombe wild-type and wee1 mutant cells. p56cdc13 has also been followed in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) cdc13 levels in wild-type cells start to rise from base line at about mid-G2, reach a peak before mitosis and then fall slowly through G1. Cells exit mitosis with appreciable levels of cdc13. (2) cdc13 levels in wee1 cells fall to zero in interphase. They also start to rise at the beginning of G2, which may be related to the absence of a mitotic size control. (3) cdc25 starts to rise later and reaches a peak after mitosis. This is not what would be expected from a simple mitotic inducer and suggests that cdc25 has an important function at the end of mitosis. (4) An upper (heavier) band of cdc25 peaks at the same time as the main band but rises and falls more rapidly. If this is a hyperphosphorylated form, its timing shows that it is most unlikely to function in the ways shown for such a form in eggs and mammalian cells. (5) Experiments with the mutant cdc10-129 and with hydroxyurea show that the initial signal to begin synthesis of cdc13 originates at Start. (6) In induction synchrony, where G2 spans across cell division, there is evidence that some events in one cycle cannot start in the previous one. (7) Revised timings are given for the times of mitosis in these cultures.

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