August 13th
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, Buscà et al. uncover a novel mechanism governing start codon selection, while Yang et al. investigate the role of the N4-acetylcytidine modification in the resistance of gastric cancer to anti-angiogenic treatment. Finally, Shin et al. analyse the effect of diphthamide loss on translation elongation fidelity.
N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2
Nucleic Acids Research, 2023
Buscà, R., Onesto, C., Egensperger, M., Pouysségur, J., Pagès, G. and Lenormand, P.
ERK proteins are involved in key cellular processes, such as proliferation and differentiation. While ERK activity is largely influenced by its double phosphorylation via MEK, the quantity of its proteins has also been shown to play a significant role in several processes, including chemoresistance. Protein production is often regulated at the level of translation, including regulation of start codon selection. With the quantity of ERK production being important in downstream processes, attention was shifted to the unusual N-terminus (Nt) of ERK, being especially enriched in alanine residues.
Here the authors used cells transfected with varying HA-tagged ERK reporters which revealed that the presence of the ERK Nt resulted in higher weight protein products. In contrast, lower weight products were associated with proteins initiated from downstream start codons, a result of leaky scanning. Results showed that the nucleotide sequence of repeating GCG is crucial to start codon selection. Replacement of these codons with alternate alanine codons significantly reduced reporter activity. These sequences must also be immediately present downstream of the start codon in question, with gaps of 1, 2, or 3 nucleotides having a significant impact on the fidelity of start codon selection. Further phylogenetic research revealed hundreds of genes with alanine-enriched N-terminals (including PABPN1), with a general favour towards more GC-rich alanine codons. Glycine codons were also enriched, although they were notably absent as the codon immediately downstream of the AUG.
N4‐Acetylcytidine Drives Glycolysis Addiction in Gastric Cancer via NAT10/SEPT9/HIF‐1α Positive Feedback Loop
Advanced Science, 2023
Yang, Q., Lei, X., He, J., Peng, Y., Zhang, Y., Ling, R., Wu, C., Zhang, G., Zheng, B., Chen, X. and Zou, B.
Gastric cancer (GC), despite advancements in treatment, remains the fourth highest cause of cancer-related deaths worldwide. Anti-angiogenic therapies have improved outcomes, but have also led to tumour hypoxia, resulting in metabolic reprogramming providing resistance to such treatments. This hypoxia activates HIF-1α, as well as the mRNA modification N4‐acetylcytidine (ac4C), although the role of such a modification in hypoxia tolerance remains unclear. NAT10, the ac4C “writer” protein, is also associated with tumour progression. While the ac4C modification is known to impact stability, the precise mechanisms of its functionality remain elusive. Here, the authors aim to unravel the impact of this modification in hypoxia and GC.
Perhaps expectedly, hypoxia in GC led to upregulation of the ac4C writer NAT10. Furthermore, stable knockdown of this gene in GC cells also resulted in lower expression of the HIF-1 pathway and genes associated with glycolysis. Seven genes, including SEPT9 (a gene the authors show to be linked to glycolysis in GC), were identified as having reduced ac4C modifications as well as reduced translational efficiency in NAT10 knockdown cells. Sites in the 3’ UTR were identified as ac4C targets, and mutations to these sites significantly reduced SEPT9 mRNA stability. Functionally, inhibition of NAT10 in conjunction with anti-angiogenic treatment significantly reduced tumour metastasis and hypoxia tolerance within tumour cells.
eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity
Nucleic Acids Research, 2023
Shin, B.S., Ivanov, I.P., Kim, J.R., Cao, C., Kinzy, T.G. and Dever, T.E.
Diphthamide (DPH) is a modified histidine found within the translation elongation factor eEF2. Its formation is catalyzed by seven highly conserved enzymes. Despite this conservation, DPH is surprisingly non-essential in yeast and in mammalian cell culture. Additionally, it serves as a target for several bacteria including Corynebacterium diphtheriae the causative agent of diphtheria. DPH interacts with the ribosomal decoding centre, and loss of DPH is also known to increase frameshifting on programmed -1 frameshifting sites. Implications that DPH may play a wider role in translational elongation are as of yet unproven. Here, the authors aimed to investigate such a potential role.
The authors found that loss of DPH did not appear to have any impact on the translocation of tRNAs in the context of codon repeats. However, it did appear to increase luciferase activity in a construct containing an accidental single nucleotide deletion in the Renilla ORF, suggesting that DPH may play a role in maintaining the proper reading frame during elongation. Further investigation revealed that the absence of DPH led to heightened frameshifting not just at programmed frameshifting sites but genome-wide, including at sites that lack slippery sequences. This increased frameshifting was detectable at -1 PRF sites, with no observable changes at +1 PRF sites. Ribosome profiling also showed that loss of DPH resulted in increased ribosome drop-off in both yeast and mammalian cells, especially on longer proteins. This was linked to termination at out-of-frame stop codons, as a result of aberrant frameshifting. The authors conclude that these results reveal a critical role for DPH in maintaining the fidelity of translation elongation and in the prevention of spurious frameshifting.