December 10th

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, we first take a look into the world of RNA modifications, as Xu et al. investigate the impact of the N4-acetylcytidine on neuropathic pain. Later, Subramanian et al. give a fascinating presentation of their latest findings into the impact of Legionella pneumophilia on the eukaryotic translational apparatus. Finally, Mao et al. develop a new ribosome profiling protocol, uncovering a potentially groundbreaking mechanism of cellular adaption to stress.

Ac4C Enhances the Translation Efficiency of Vegfa mRNA and Mediates Central Sensitization in Spinal Dorsal Horn in Neuropathic Pain

Advanced Science, 2023

Xu, T., Wang, J., Wu, Y., Wu, J.Y., Lu, W.C., Liu, M., Zhang, S.B., Xie, D., Xin, W.J. and Xie, J.D.

Neuropathic pain treatments often lack efficacy, likely due to a lack of understanding of the basic underlying mechanisms. Recently, emerging evidence suggests that the epitranscriptome may play a role in such conditions. N4-acetylcytidine (ac4C) is a conserved RNA modification with known roles in mRNA stability and translation, and is implicated in varying neurological diseases. It’s “writer”, N-acetyltransferase 10 (NAT10) is, however, uncharacterised with regard to neuropathic pain. Vascular endothelial growth factor A (VEGFA) is itself upregulated following nerve injury. Here, the authors investigated if there was any overlap between the ac4C modification and VEGFA-associated neuropathic pain.

Here, ribosome sequencing (Ribo-seq) and other molecular techniques were used to analyze gene expression and translation efficiency. In a gene set enrichment analysis, members of the VEGF pathway scored among the highest in terms of ac4C modifications following nerve injury. VEGFA itself displayed increased enrichment of ac4C modifications within its 3’ UTR. Despite previous research indicating ac4C increases mRNA stability, the increase in VEGFA protein was instead attributed to increased VEGFA translational efficiency. NAT10 expression also concomitantly increased following nerve injury, and displayed enrichment at the newly identified VEGFA ac4C modification site. Additionally heterogeneous nuclear ribonucleoprotein K (HNRNPK) was identified as a co-factor in NAT10 mediated VEGFA ac4C modification, with its knockdown reducing the level of such a modification. This work helps advance the identification of novel targets for the development of effective treatment for neuropathic pain induced by nerve injury.

A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response

Nature Cell Biology, 2023

Subramanian, A., Wang, L., Moss, T., Voorhies, M., Sangwan, S., Stevenson, E., Pulido, E.H., Kwok, S., Chalkley, R.J., Li, K.H. and Krogan, N.J.

Legionella pneumophilia, the bacterium responsible for Legionnaires’ disease, utilises a type IV secretion system to deliver around 300 protein effectors into the host cells. A number of these, such as Lgt1-3 and LegK4, are known to interfere in translation elongation, targeting factors such as eEF1A. Others, such as SidI, have unknown mechanisms of action. Legionella infection typically results in either cell death or strong activation of the p38 and JNK kinases. Separately, recent evidence suggests ribosome collisions induces activation of the integrated stress response (ISR) and the ribotoxic stress response (RSR). Here, the authors aim to investigate any commonality between these cellular responses, with a focus on the under-investigated protein, SidI.

In testing of the redundancy of L. pneumophilia effectors in a cell-free translation system, SidI was found to be the most potent in arresting translation. Further investigation revealed a strong cluster of native SidI interactors to be associated with the protein synthesis apparatus, precipitating eEF1A and tRNA ligases. Structural analysis suggested that SidI possessed a feature similar to proteins known to act as tRNA mimics, as well as a second feature resembling a known mannosyl transferase domain. Functionally, these domains were later shown to target the ribosome and glycosylate it respectively. Together with ribosome profiling, this was demonstrated to stall elongating ribosomes, and induce the RSR, leading to accumulation of ATF3 protein, ultimately regulating cell fate.

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation

Nature Structural and Molecular Biology, 2023

Mao, Y., Jia, L., Dong, L., Shu, X.E. and Qian, S.B.

Eukaryotic translation initiation involves a complex of factors, together coordinating ribosome loading and start codon selection. The transition from translation initiation to elongation is not fully understood. Ribosome profiling captures a snapshot of translation, and its use has revealed a large proportion of reads originating out-of-frame. Out-of-frame reads typically result from either frameshifting events (be they programmed or non-programmed), or alternative initiation sites out-of-frame from the canonical start. Here, the authors investigate the transition between initiation and elongation with a novel ribosome profiling technique, named Ezra-Seq.

They demonstrate increased resolution with this technique compared to previous iterations. While out-of-frame footprints are reduced with this technique, a significant proportion remain. Metagene analysis uncovers an enrichment of out-of-frame reads at the codon immediately following the beginning of coding regions (CDS). Interestingly, strong start codons tended to have a greater comparative proportion of these reads, compared to weaker starts. Fluorescent reporter assays demonstrated that reduced codon optimality, often found in the early regions of the CDS, led to increased frameshifting, and that initiating ribosomes were more susceptible to such frameshifting. Using intricately designed reporters with polysome-seq, they were able to localise frameshifting events specifically to the start codons. Further context orientated investigation revealed that a U at the +5 position suppressed such frameshifting. Interestingly, cells lacking eIF5B demonstrated start codon-associated frameshifting (SCARF). Furthermore, cell starvation induces SCARF, through eIF5B degradation, leading to amino acid scavenging from the quickly polyubiquitinated out-of-frame proteins that result.

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