Background. Translational and rapid RNA abundance control is important in all life but remains a challenge to accurately quantify. Messenger (m)RNA stability has recently been tightly linked with the translational efficiency. At the same time, covalent non-templated modifications to the RNA nucleotides, collectively known as the epitranscriptome, have been shown to alter translational rates and stability of the mRNA. The interlink between the translation, turnover and RNA modifications needs to be explored to fully expose the mRNA function. Yet, no study has attempted to investigate these processes in the same system transcriptome-wide.
Aims and Approach. Here we for the first time use an unbiased, unsupervised approach to reveal connections between the translation rates, positions and stoichiometry of the major modifications in mRNA, and the mRNA degradation rates. We use accurate translational rate predictions derived from short-read rapid crosslinking-based enhanced translation complex profile sequencing (eTCP-seq) and Stochastic Translation Efficiency (STE)1 AI in naive and glucose-stressed yeast cells. Concurrently for the same cell material, we employ direct RNA sequencing (DRS) and in-house tools for modification (CHEUI/SWARM)2 and RNA degradation (INDEGRA)3 measurement reliant on accurate DRS data.
Results and Conclusions. We demonstrate diverse classes of mRNAs strongly associating with the similar changes in translational rates, particularly translation initiation frequency and stalling profiles, mRNA turnover and the N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (pU) and N4-acetylcytidine (ac4C) stoichiometry patterning along transcript regions. Our data for the first time reveal a multifaceted complexity of acute stress response control at the RNA level, and provide a direct proof of the interlink between the translation initiation/elongation, epitranscriptome marks and RNA turnover. Our results enable new applications in dissecting cell states in disease pathophysiology and drug development by the complex RNA response, and will facilitate the design of next-generation synthetic biology constructs and mRNA-based therapeutics.