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

Abstract

Cells 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..

PABP prevents the untimely decay of select mRNA populations in human cells

“…We therefore sought to determine whether changes to mRNA decay rates could explain PABP‐dependent changes in mRNA levels. To this end, we first used published data of mRNA half‐lives in a human cell line (human umbilical vein endothelial cells) and compared it to PABP‐dependent changes in mRNA levels (Tiana et al , 2020). Indeed, mRNAs whose abundance decreased following PABP depletion were reported to have significantly longer half‐lives relative to mRNAs whose steady‐state levels increased following loss of PABP (Fig 5A).…”

Hypoxia re-programs 2′-O-Me modifications on ribosomal RNA

“…A major difference between Tiana et al., and this manuscript is the length of hypoxic incubation. Tiana et al., use significantly shorter periods of hypoxia (8–16 hr) whereas work presented here uses 72 hr ( Tiana et al., 2020 ). However, an interesting likelihood emerges that integrates these apparent discrepancies.…”

Formal meta-analysis of hypoxic gene expression profiles reveals a universal gene signature and cell type-specific effects

“…Since the meta-analyses included experiments done at relatively short exposure times (27% of the subsets correspond to exposure times ranging 6/21 from 1-12h), it could be argued that the smaller effect size observed for repressed genes is a consequence of short-time experiments failing to detect the effect on mRNA levels due to their half-life. To test this hypothesis, we repeated the meta-analyses selecting only subsets corresponding to treatments of 24-48h, significantly longer than the median half-life of 5.7 h observed for human mRNAs under hypoxia [20]. As shown in figure 3D left panel, both distributions show a small shift toward higher absolute effect size values, but the difference between them remains unaltered.…”