May 5th, 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, Popper et al. examined global translation kinetics across various cell types using timed ribosome runoff, proteomics, and electron microscopy. Misra et al. investigated GCN2 activation mechanisms. Finally, a study by O’ Connor et al. highlights the role of translation in the Lactococcus cremoris during sk1 bacteriophage infection.

Ribosome inactivation regulates translation elongation in Neurons

Journal of Biological Chemistry, 2024

Bastian Popper, Martina Bürkle, Giuliana Ciccopiedi, Marta Marchioretto, Ignasi Forné, Axel Imhof, Tobias Straub, Gabriella Viero, Magdalena Götz, and Rico Schieweck

Translation, a complex process, is tightly regulated by factors like RNA-binding proteins, tRNAs, codon usage, and RNA structures, impacting translational efficiency and protein levels. Ribosomes, once viewed as mere translators, now serve as active regulators. Neurons, with intricate shapes and diverse stimuli, require precise translational control. Ribosome speed, influenced by various factors including tRNA levels and elongation factors, varies between transcripts and tissues. Dysregulated elongation can lead to protein misfolding and diseases. Using timed ribosome runoff, proteomics, and electron microscopy, this study examined global translation kinetics across various cell types.

In this study, authors investigated global translation kinetics using harringtonine (HRN), which immobilizes initiating ribosomes to map translation start sites and study ribosome elongation speed. Various cell types were examined including human neural stem cells (hNSCs), immature and mature rat cortical neurons (RCNs), human induced pluripotent stem cells (IPSCs) differentiated into neurons, and cultured astrocytes. Polysome profiling revealed a decline in polysomes and an increase in monosomes with HRN treatment, indicating ribosome runoff. Polysome rate constants (kP) and polysome-to-monosome ratios (P/M(0)) varied between cell types and revealed that mature RCNs exhibited higher kP values than HEK cells, indicating faster ribosome speeds. Neuronal maturation was associated with increased ribosome speed, suggesting its importance in nerve cell development. Neuronal excitation triggers increased ribosome inactivation in an eEF2-dependent manner, suggesting a dynamic regulatory mechanism. These results highlight differences in ribosomal speeds across cell types and emphasize the role of translational speed in neuronal development.

These findings offer insights into translational remodelling and have implications for developmental brain disorders characterized by aberrant translation. These discoveries hold significant implications for developmental brain disorders marked by irregularities in translation, among other factors.

Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions

Nucleic Acids Research, 2024

Jagannath Misra, Kenneth R Carlson, Dan F Spandau, and Ronald C Wek

The integrated stress response (ISR) orchestrates cellular adaptation to various disturbances by phosphorylating eIF2α, reducing protein synthesis, and activating stress-adaptive gene expression, including ATF4. The eIF2 kinase GCN2 initiates ISR, responding to amino acid depletion and other stresses. GCN2 activation can occur through monitoring uncharged tRNAs or stalled ribosomes. Findings from this study underscore multiple mechanisms of GCN2 activation in response to different stresses, involving both direct tRNA engagement and ribosomal collisions.

The study investigated whether ZAK-deleted keratinocytes exhibit translational control similar to wild-type (WT) cells upon UV irradiation. Both WT and ZAK-deleted cells showed a sharp decrease in polysome to monosome ratio after UV-B exposure, indicating reduced translation initiation. This suggests that ZAK is not required for GCN2 activation and translational control in keratinocytes exposed to UV. Moreover, UV-B-induced GCN2 activation was observed even in the presence of puromycin, indicating that ribosome collision is not necessary for GCN2 activation by UV-B irradiation. Similarly, UV-B failed to activate a mutant version of GCN2, indicating that both ZAK and ribosome collisions are dispensable for GCN2 activation by UV-B irradiation. Results showed that reduced levels of translation elongation inhibitors like anisomycin can lead to ribosome collisions, triggering ZAK activation, subsequently initiating GCN2 within the ISR.

This study reveals ribosomal collisions essential for GCN2 activation under translational inhibitors, while tRNA deacylation activates GCN2 directly. UV-induced GCN2 activation involves decreased amino acids and increased uncharged tRNAs, with ribosome collisions dispensable. This highlights diverse stress induced GCN2 activation mechanisms.

Ribosome profiling reveals downregulation of UMP biosynthesis as the major early response to phage infection

Microbiology Spectrum, 2024

PBF O’Connor, J Mahony, E Casey, PV Baranov, D van Sinderen, and MM Yordanova

 

Bacteria have developed a variety of defence strategies to protect themselves against bacteriophage (phage) attacks. These defences are linked to genetic responses that occur when a bacteriophage infects a host, influencing the infection’s outcome. This study highlights the role of translation in the Gram-positive bacterium Lactococcus cremoris, during sk1 bacteriophage infection.  In this research, ribosome profiling was employed to track protein production in the early phases of sk1 bacteriophage infection in L. cremoris.

The authors observed shutdown in nucleotide import and salvage pathways, coupled with the downregulation of various pyr operons, indicates a strategic limitation of nucleotide availability as a potential anti-phage defence mechanism.The research also suggests that in L. cremoris, phage infection may trigger an increase in UMP levels, leading to PyrR-UMP-mediated downregulation of pyr operons, thereby depleting the cell’s dNTPs to disrupt phage DNA replication. However, despite these defensive measures, sk1 phage remains highly virulent, indicating evolved mechanisms to bypass host defences.

Interestingly, the study also notes an upregulation of genes associated with protein synthesis and chaperones during infection, potentially benefiting phage propagation. This could imply a phage-induced regulatory shift in the host, favouring rapid production over translational fidelity, which might align with a phage’s higher tolerance for translational errors.

Moreover, the application of ribosome profiling (Ribo-seq) in this study has provided valuable insights into gene expression alterations during phage infection, validating previously annotated pseudogenes and identifying the translation of loci previously considered non-functional. This reveals a nuanced layer of gene expression in L. cremoris, with implications for understanding phage-host dynamics and the evolutionary adaptations of both entities. The translation of pseudogenes, some resulting in truncated proteins, opens new avenues for understanding their roles and potential functions post-infection.

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