November 12th
Recent Publications Harnessing the Power of Translatomics.
Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.
In this week’s Sunday papers, Ferguson et al. (2023) present a simplified method to improve ribosome footprint enrichment in comparison to ribosomal RNA and decreased sequencing bias. Millius et al. (2023) used ribosome profiling of circadian-entrained mice to investigate post-transcriptional control of circadian rhythms. Lastly, Rahaman et al. (2023) discovered that the length of the mRNA coding region determines the increase in mRNA stability in yeast.
Streamlined and sensitive mono-and diribosome profiling in yeast and human cells
Nature Methods, 2023
Ferguson, L., Upton, H.E., Pimentel, S.C., Mok, A., Lareau, L.F., Collins, K. and Ingolia, N.T
Through a transcriptome-wide study of ribosome occupancy, as revealed by the sequencing of messenger RNA snippets shielded by ribosomes, ribosome profiling has shown a variety of control and perturbations of translation. From a technical standpoint, it is difficult to generate ribosome footprints and convert them into sequencing libraries, while the procedure is also sensitive to biases which may skew the representation of physiological ribosome occupancy. Here, the authors overcome these obstacles by using P1 nuclease (a sequence-independent, single-strand nuclease) instead of RNase I to create ribosome footprints and by using ordered two-template relay (OTTR) in place of RNA ligation to prepare sequencing libraries in a single tube while incorporating adaptors through reverse transcription.
They present a simplified method to improve ribosome footprint enrichment in comparison to ribosomal RNA and decreased sequencing bias. P1 nuclease footprinting and OTTR library synthesis in humans and yeast identified many configurations of contiguous, colliding ribosomes that cause ribosome and/or mRNA turnover through ribosome quality control (RQC) pathways. This method also identified the ribosomal stalling caused by impaired translation. They claim that their refined techniques for creating and capturing mRNA footprints and removing gel electrophoresis when recovering RPFs yield a richer translatome profile with minimal input and fewer technological difficulties.
Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep
Proceedings of the National Academy of Sciences, 2023
Millius, A., Yamada, R.G., Fujishima, H., Maeda, K., Standley, D.M., Sumiyama, K., Perrin, D. and Ueda, H.R.
The synthesis and degradation of many mammalian proteins follow circadian cycles, and a number of these rhythms are modified post-transcriptionally. The authors used ribosome profiling to investigate posttranscriptional control of circadian rhythms. They measured RNA translation in the liver over a 24-hour period from circadian-entrained mice transferred to constant darkness conditions and also compared ribosome binding levels to protein levels for 16 circadian proteins. They report significant discrepancies between the levels of ribosome binding and protein, as well as delays between peak ribosome binding and peak protein abundance. They discovered that ribosomes bind extensively to circadian mRNAs’ upstream open reading frames (uORFs), which include the fundamental clock gene Period2 (Per2).
A decrease in ribosome binding in the main coding region and a decrease in the expression of synthetic reporter constructs were linked to an increase in uORFs in the 5′UTR. The expression of luciferase and fluorescence reporters was elevated in 3T3 cells and PER2:LUC MEF cells when the Per2 uORF was mutated. When compared to wild-type mice, mice with a mutation in the Per2 uORF showed greater expression of Per2 mRNA, improved ribosome binding to Per2, and decreased overall sleep time. These findings imply that uORFs influence mRNA post-transcriptionally, which may have an effect on sleep and physiological cycles.
Polysome propensity and tunable thresholds in coding sequence length enable differential mRNA stability
Science Advances, 2023
Rahaman, S., Faravelli, S., Voegeli, S. and Becskei, A.
The translation and half-life of mRNAs both rise in proportion to the optimal codons, suggesting a close relationship between codon-dependent differential translation and degradation. However, several mRNA classes have half-lives that the standard model is unable to account for. For instance, when mRNAs are sorted based on the number of ribosomes translating them, it has been observed that mRNAs with short coding sequences (CDS) have shorter half-lives than predicted, and are predominant in the monosomal fraction. Because the polysome profiles of optimal codon-rich and optimal codon-poor mRNAs are similar, it is difficult to investigate this mechanism in detail. Consequently, regardless of their optimal codon content, mRNAs have comparable ribosome densities.
Here, the authors discovered that the length of the mRNA coding region determines the increase in mRNA stability in yeast. Codon optimality does not influence the stability of mRNAs below a critical length, despite the fact that they can be translated into small peptides and proteins with high efficiency. Over this critical length, the mRNAs’ propensity to be associated with polysomes increases similarly, and codon optimality–dependent differential mRNA stability manifests itself in a switch-like manner. The untranslated regions (UTR) have the ability to tune this threshold length. A portion of these UTRs have the ability to destabilize mRNAs without decreasing translation, which helps regulate the oscillatory expression of cell cycle genes’ amplitude.