Ribosome stacking during translation of messenger (m)RNA has long been observed and recently re-discovered in ribosome profiling, being attributed to peptide elongation slowdown. In contrast to this dogma, we propose and, using enhanced translation complex profile sequencing, confirm co-localisation of ribosomes on mRNA resulting from the diffusional dynamics. We further present a comprehensive view on the mechanisms and types of ribosomal co-localisations on mRNA. We demonstrate that the co-localised ribosomes (such as disomes) can be of a different origin. Some co-localised ribosomes are related to the well-accepted translation elongation delays on mRNA. Others are reflective of the mRNA spatial arrangement in polysomes and descend from individual molecular co-localisation events.
We find that certain stochastically co-localised ribosomes are linked to the translation initiation efficiency and provide a robust variable to model specific protein output from mRNA. Employing unbiased machine learning and the stochastic signal, together with other variables derived from an enhanced translation complex profile sequencing (TCP-seq; Archer et al. 2016 Nature, Shirokikh et al. 2017 Nature Protocols, Janapala et al. 2021 JoVE), we demonstrate accurate prediction of the absolute translation output in the form of Stochastic Translation Efficiency (STE) measure (Horvath et al. 2022 bioRxiv). Using STE, it is possible to rank mRNAs by the ‘power’ of translation control elements across transcripts in a single experiment or between different conditions. STE does not use bias-inducing normalisation to the RNA abundance or signals of different types and relies on self-normalised signal pairs.
Applying STE to the prototypical example of translational control during yeast response to glucose depletion, we find that glucose stress results in a response that is more complex than previously thought, exhibiting a high degree of selective translational control that acts towards both, suppression and activation of different mRNAs. Unexpectedly, we uncover elevated initiation rate for the many of ‘mid-power’ mRNAs under the stress. We suggest STE use for dissecting finer dynamics of translation and elucidation of very rapid cell responses inaccessible to the other approaches.