Oral Presentation Australasian RNA Biology and Biotechnology Association 2024 Conference

Accurate protein biosynthesis rates link with the epitranscriptome and RNA degradation to reveal the function of mRNA in acute nutrient stress (108981)

Orlane Rossini 1 , Shafi Mahmud 2 , Katrina Woodward 2 , Thomas Preiss 2 3 , Nikolay E. Shirokikh 2 , Alice Cleynen 1 2
  1. The National Centre for Scientific Research (CNRS), Université de Montpellier, Montpellier, France
  2. Division of Genome Sciences and Cancer, The John Curtin School of Medical Research, and The Shine-Dalgarno Centre for RNA Innovation, The Australian National University, Canberra, ACT, Australia
  3. Victor Chang Cardiac Research Institute, Sydney, NSW , Australia

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.

  1. Attila Horvath, Yoshika Janapala, Katrina Woodward, Shafi Mahmud, Alice Cleynen, Elizabeth E Gardiner, Ross D Hannan, Eduardo Eyras, Thomas Preiss, Nikolay E Shirokikh "Comprehensive translational profiling and STE AI uncover rapid control of protein biosynthesis during cell stress" Nucleic Acids Research 2024; gkae365, https://doi-org.virtual.anu.edu.au/10.1093/nar/gkae365
  2. P Acera Mateos, A J Sethi, A Ravindran, A Srivastava, K Woodward, S Mahmud, M Kanchi, M Guarnacci, J Xu, Z W S Yuen, Y Zhou, A Sneddon, W Hamilton, J Gao, L M Starrs, R Hayashi, V Wickramasinghe, K Zarnack, T Preiss, G Burgio, N Dehorter, N E Shirokikh & E Eyras "Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications" Nature Communications 2024; 15: 3899
  3. Alice Cleynen, Agin Ravindran, Aditya Sethi, Bhavika Kumar, Shafi Mahmud, Katrina Woodward, Tanya Javaid, Stéphane Robin, Eduardo Eyras, Nikolay Shirokikh "High-Accuracy RNA Integrity Definition for Unbiased Transcriptome Comparisons with INDEGRA" https://github.com/Arnaroo/INDEGRA