Expression profiling by high throughput sequencing
Summary
The prevailing model postulates that complete cytoplasmic polyadenosine tail (pA-tail) deadenylation is essential for initiating mRNA decapping and subsequent degradation. To investigate this, we conducted direct RNA sequencing of yeast mRNAs derived from steady-state and stress condition chase experiments. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile pA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNA, which appeared consistent within functional transcript groups and associated with to codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40 % of the transcriptome, singled out this transcript group. While RPG mRNA deadenylation and decay accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating decapping onset, while also demonstrating complex relations between these processes.
Overall design
We implemented Nanopore Direct RNA Sequencing to we experimentally model RNA deadenylation and decay rates under steady-state and stress conditions using: (1) the Anchor-Away system, to deplete the main cellular export factor, Mex67, thus rapidly inducing massive nuclear degradation of newly-synthesized mRNAs, (2) depletion of the decapping enzyme Dcp2, Xrn1 5'-3' exonuclease, or Pab1 poly(A)-binidng protein using the auxin-inducible degron (AID) system, (3) a heat stress chase, (4) a thiolutin chase.
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