scholarly journals Metabolic labeling of RNA using multiple ribonucleoside analogs enables the simultaneous evaluation of RNA synthesis and degradation rates

2020 ◽  
Vol 30 (10) ◽  
pp. 1481-1491
Author(s):  
Kentaro Kawata ◽  
Hiroyasu Wakida ◽  
Toshimichi Yamada ◽  
Kenzui Taniue ◽  
Han Han ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Metabolic Labeling ◽  
Simultaneous Evaluation ◽  
Degradation Rates ◽  
Synthesis And Degradation

scholarly journals Metabolic labelling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels

2019 ◽  
Author(s):  
Maria Tiana ◽  
Clara Galiana ◽  
Miguel Ángel Fernández-Moreno ◽  
Benilde Jimenez ◽  
Luis del Peso
Keyword(s):  
Rna Synthesis ◽  
Environmental Changes ◽  
Hypoxia Inducible Factor ◽  
Rna Degradation ◽  
Decay Rates ◽  
Specific Gene ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Degradation Rates ◽  
Rna Levels

ABSTRACTCells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that the changes in transcription rates induced by hypoxia are the major determinant of RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect was mimicked by forced activation of the Hypoxia Inducible Factor pathway and prevented by its interference. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.

scholarly journals Metabolic labeling of RNA using multiple ribonucleoside analogs enables simultaneous evaluation of transcription and degradation rates

2020 ◽  
Author(s):  
Kentaro Kawata ◽  
Hiroyasu Wakida ◽  
Toshimichi Yamada ◽  
Kenzui Taniue ◽  
Han Han ◽  
...  
Keyword(s):  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Rna Degradation ◽  
Functional Enrichment ◽  
Metabolic Labeling ◽  
Simultaneous Evaluation ◽  
Degradation Rates ◽  
Relationship Of ◽  
External Stimuli ◽  
The Relationship

SummaryGene expression is one of the factors determining cellular conditions, and its level is determined by the balance between transcription and RNA degradation. However, it has been not well elucidated how transcription and degradation are related to each other. To elucidate the relationship of them, methods enabling simultaneous measurement of transcription and degradation are required. Here, we report the development of “Dyrec-seq” to evaluate transcription and RNA degradation rates simultaneously, by using 4-thiouridine and 5-bromouridine. Using Dyrec-seq, we quantified the transcription and degradation rates of 4,702 genes in HeLa cells. Functional enrichment analysis showed that the transcription and degradation rates of genes are actually determined by the genes’ biological functions. Comparison of theoretical and experimental analysis results revealed that the amount of RNA was determined by the ratio of transcription to degradation rates, while the rapidity of responses to external stimuli was determined only by the degradation rate. This study emphasizes that degradation as well as transcription is important in determining the behavior of RNA.

scholarly journals Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 18O2 labelling

1986 ◽  
Vol 240 (2) ◽  
pp. 431-435 ◽  
Author(s):  
J A Molnar ◽  
N Alpert ◽  
J F Burke ◽  
V R Young
Keyword(s):  
Gas Chromatography Mass Spectrometry ◽  
Collagen Turnover ◽  
Growing Rats ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Degradation Rates ◽  
Labelling Procedure ◽  
Skin Biopsies ◽  
Synthesis And Degradation

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.

scholarly journals Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells

1978 ◽  
Vol 176 (3) ◽  
pp. 933-941 ◽  
Author(s):  
W T Melvin ◽  
H M Keir
Keyword(s):  
Cell Growth ◽  
Dna Synthesis ◽  
Protein Content ◽  
Rna Synthesis ◽  
Step Down ◽  
Rrna Degradation ◽  
Synthesis And Degradation

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after ‘step-down’ rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after ‘step-down’. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.

Kinetic analysis of segmentation gene interactions in Drosophila embryos

Development ◽  
1999 ◽  
Vol 126 (7) ◽  
pp. 1515-1526 ◽  
Author(s):  
A. Nasiadka ◽  
H.M. Krause
Keyword(s):  
Target Genes ◽  
Vast Number ◽  
Rate Limiting Step ◽  
Degradation Rates ◽  
Time Required ◽  
Potential Interactions ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Synthesis And Degradation ◽  
Drosophila Embryos

A major challenge for developmental biologists in coming years will be to place the vast number of newly identified genes into precisely ordered genetic and molecular pathways. This will require efficient methods to determine which genes interact directly and indirectly. One of the most comprehensive pathways currently under study is the genetic hierarchy that controls Drosophila segmentation. Yet, many of the potential interactions within this pathway remain untested or unverified. Here, we look at one of the best-characterized components of this pathway, the homeodomain-containing transcription factor Fushi tarazu (Ftz), and analyze the response kinetics of known and putative target genes. This is achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, we find that both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (<10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes are analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy.

scholarly journals Contribution of whole-body protein synthesis to basal metabolism in layer and broiler chickens

1987 ◽  
Vol 57 (2) ◽  
pp. 269-277 ◽  
Author(s):  
T. Muramatsu ◽  
Y. Aoyagi ◽  
J. Okumura ◽  
I. Tasaki
Keyword(s):  
Protein Synthesis ◽  
Broiler Chickens ◽  
Whole Body ◽  
Synthesis Rate ◽  
Body Protein ◽  
Fractional Synthesis Rate ◽  
Degradation Rates ◽  
Fractional Synthesis ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

1. The effect of starvation on whole-body protein synthesis and on the contribution of protein synthesis to basal metabolic rate was investigated in young chickens (Expt 1). Strain differences between layer and broiler chickens in whole-body protein synthesis and degradation rates were examined when the birds were starved (Expt 2).2. In Expt 1, 15-d-old White Leghorn male chickens were used, while in Expt 2 Hubbard (broiler) and White Leghorn (layer) male chickens at 14 d of age were used. They were starved for 4 d, and heat production was determined by carcass analysis after 2 and 4 d of starvation. Whole-body protein synthesis rates were measured on 0, 2 and 4 d of starvation (Expt 1), and on 0 and 4 d of starvation (Expt 2).3. The results showed that starving reduced whole-body protein synthesis in terms of fractional synthesis rate and the amount synthesized. Whole-body protein degradation was increased by starvation both in terms of fractional synthesis rate and the amount degraded on a per kg body-weight basis.4. Reduced fractional synthesis rate of protein in the whole body was accounted for by reductions in both protein synthesis per unit RNA and RNA:protein ratio.5. In the fed state, whole-body protein synthesis and degradation rates, whether expressed as fractional rates or amounts per unit body-weight, tended to be higher in layer than in broiler chickens. In the starved state, the difference in the rate of protein synthesis between the two strains virtually disappeared, while the degradation rates were higher in layer than in broiler birds.6. Based on the assumed value of 3.56 kJ/g protein synthesized (Waterlow et al. 1978), the heat associated with whole-body protein synthesis in the starved state was calculated to range from 14 to 17% of the basal metabolic rate with no strain difference between layer and broiler chickens.

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