July 7th, 2024
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, Ero et al. showed that the formation of large ribosomal subunit (50S) of E. coli was essential for its ribosome biogenesis, contributing to antibiotic development. Hacisuleyman et al. presented that uORF:eIF4G2 binding improved synaptic plasticity and dendritic function following neuronal depolarisation. Bicknell et al. stated that moderate ribosome loads, good codon optimality and longer elongation rates maximised total protein output in the field of mRNA therapeutic development.
Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli
Nucleic Acids Research, 2024
Ero, R., Leppik, M., Reier, K., Liiv, A. and Remme, J.
Post-transcriptional modification of ribosomal RNA (rRNA) is an important step during ribosome synthesis. The modification is essential during ribosome assembly and introduces various changes to the chemical properties of the rRNA. The rRNA modifications, catalysed by modification enzyme (ME), occurs in the critical regions of rRNA that interact with ribosomal proteins and other rRNA segments to stabilise the structure and function of the ribosome.
Ribosome profiling in this study serves as a powerful tool to dissect the molecular underpinnings of ribosome assembly, illustrating the indispensable roles of rRNA modifications. The authors designed to knock out the E. coli strains with several ME. Through ribosome profiling analysis, these mutants exhibited defects in ribosome assembly, particularly in the formation of the large ribosomal subunit (50S). While single ME deletions did not significantly disrupt ribosomal function, the study showed that the ribosome assembly was significantly impaired by the deletion of 10 selected ME (Δ10).
Mechanistic studies within the paper reveal that certain methylations and pseudouridylations facilitate the late stages of 50S subunit assembly by promoting interdomain interactions within the rRNA. For instance, the methylation of a specific nucleotide (Um2552) by the enzyme RlmE was found to be critical for these interactions. The absence of specific rRNA modifications disrupts the late stages of 50S subunit assembly, leading to incomplete or defective ribosomal subunits. Overall, the research improved our understanding on the essential roles of the large ribosomal subunit (50S) of the E. coli in ribosome biogenesis, shedding light on the antibiotic development.
Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding
Nature Neuroscience, 2024
Hacisuleyman, E., Hale, C.R., Noble, N., Luo, J., Fak, J.J., Saito, M., Chen, J., Weissman, J.S. and Darnell, R.B.
The paper explores how neuronal activity influences protein synthesis in dendrites, a process crucial for learning and memory. In this study, the authors employed advanced techniques such as dendritically targeted proximity labelling, crosslinking immunoprecipitation, ribosome profiling, and mass spectrometry to monitor changes in dendritic translation following neuronal depolarisation. Hacisuleyman et al. labelled dendritic TurboID with ribosome profiling to identify postsynaptic translatome through deep sequencing.
Depolarisation of primary cortical neurons with KCl or the glutamate agonist DHPG swiftly alters dendritic protein expression. This reprogramming shows a weak correlation between changes in dendritic mRNAs and the proteins produced. Through ribosome profiling analysis, a subset of pre-localized mRNAs exhibited increased translation of upstream open reading frames (uORFs) and their downstream coding sequences upon depolarization. This increase facilitates localized production of proteins involved in long-term potentiation, cell signaling, and energy metabolism. The non-canonical translation initiation factor eIF4G2 is phosphorylated and recruited during this activity-dependent translation. The translated uORFs are sufficient to induce eIF4G2-dependent translational control, linking neuronal activity to local dendritic remodelling.
These findings reveal a novel mechanism by which neuronal activity controls localised protein synthesis in dendrites, highlighting the importance of eIF4G2 and uORFs in synaptic plasticity and neuronal function.
Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay
Cell Reports, 2024
Maximising protein output per delivered mRNA molecule is desirable in mRNA therapeutic. Higher polysome density would maximise total protein output from a therapeutic mRNA, however, previous studies showed that the mRNAs in the largest polysomes yielded lower total protein output due to their significantly shorter half-lives. Bicknell et al. conducted the study to understand the impact of CDS design, which affects the translation initiation and elongation rates, on mRNA lifetimes. Polysome profiling was employed to quantify the absolute number of ribosomes per mRNA, and to determine the rate at which mRNAs shifted to lighter polysome fractions as ribosomes disassociated.
mRNA decay rates varied across different sequence groups. The study identified that sequence variants with different physical mRNA lifetimes was the limiting factor on protein synthesis. Translation-dependent and translation-independent mechanisms can also contribute to the variability of the mRNA decay rates. Translation initiation inhibitor lactimidomycin (LTM) was used to investigate the translation-dependent and -independent decay rates. It showed that the decay of high codon optimality/high-structure mRNA was significantly independent of translational mechanism. Translation on non-optimal codon and rapid initiation boosted ribosome loading. Designing CDS to balance codon usage and secondary structures extended mRNA lifetime, further increasing protein expression. A deadenylation suppressor, 3′ inverted deoxythymidine (InvdT), was added to the cells to block decapping or exosome-mediated degradation. This modification was proven to be beneficial to improve the mRNA half-life.
Overall, the research suggested that the minimal translation-dependent mRNA decay can be achieved by achieving moderate ribosome loads and longer mRNA half-lives. This finding drove interests in the mRNA therapeutic development by potentially minimising ribosome load.