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Accession IconSRP157719

Metabolic labeling of RNAs uncovers hidden features and dynamics of the Arabidopsis thaliana transcriptome [ERIC]

Organism Icon Arabidopsis thaliana
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Technology Badge IconIllumina HiSeq 3000

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We provide evidence that 5-EU metabolic labeling of Arabidopsis RNAs can be used for pulse-chase experiments, which allowed us to determine Arabidopsis RNA half-lives genome-wide without chemical inhibition of transcription. Similar to BRIC-Seq, we performed 5-EU IP chase (ERIC)-Seq in seedlings of A. thaliana. Hierarchical clustering of gene expression values allowed us to define at least five clusters of mRNAs that exhibited distinct degradation kinetics. To determine the RNA half-lives of each group, we applied an exponential decay model and a locally weighted scatterplot smoothing (LOWESS) and calculated the time where the concentration was halved. It became obvious that the half-lives determined by ERIC-Seq are much shorter than the ones determined after treatment with cordycepin or actinomycin D, respectively. We found that genes belonging to cluster A exhibiting the shortest half-lives are enriched in genes transcribed into non-coding RNAs, stress-related genes, genes involved in hormonal pathways and the metabolism of polyamines, which are also involved in stress responses. On the contrary, genes from cluster E, which have the longest half-lives, are specifically enriched in genes responsible for mitochondrial and plastic functions as well as primary metabolism (such as the TCA cycle) Overall design: RNA was metabolically labeled with 5-ethynyl uridine for 24h, then the seedlings were transferred to media containing a high concentration of uridine to chase the 5-ethynyl uridine out. Samples were taken subsequently at 0h, 1h, 2h, 6h, 12h and 24h after end of labeling period. A known concentration of a EU containing RNA was used as spike-in for normalization. The poly(A) fraction of total RNA was used for NGS library preparation. Samples were taken in triplicates; an sample of timepoint zero hours was taken as reference.
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