Fig. 4: Ribo-seq reveals defective decoding of codons with adenosine in the third position following polyamine depletion.
From: Reprogramming neuroblastoma by diet-enhanced polyamine depletion
a, For functional evaluation of translation, tumours were lysed in the presence of a translation inhibitor for preparation of RNA-seq and Ribo-seq libraries. Ribosome-protected RNA fragments were isolated and sequenced to assess translation. b, Normalized ribosome depth at positions encoding three or more consecutive proline residues. Decoding of these polyproline tracts is affected by combining DFMO (1% in the drinking water) with proline and arginine-free diet. c, Proline translation defects are codon-specific. Relative ribosome density centred around proline codons across treatment groups relative to CD (zero line). Left, density of ribosomes at polyproline tracts. Right, codon occupancy on proline codons outside of polyproline tracts. Increased occupancy manifests at CCA and less at CCC. d, Codons with adenosine in the third position show specific translation defects induced by the combined ProArg-free diet plus DFMO treatment compared with CD diet when comparing the transcriptome-wide relative ribosome occupancy. Codons that require tRNAs with modifications at position 34 for decoding are highly enriched in codon pausing following ProArg-free diet plus DFMO treatment compared with CD. Relative pausing of codons in the ribosomal P site. GalQ, galactosyl-queuosine; I, inosine; manQ, mannosyl-queuosine; mchm5U, 5-methoxycarbonyl-hydroxymethyluridine; mcm5s2U, 5-methoxycarbonylmethyl-2-thiouridine; ncm5U, 5-carbamoylmethyluridine; Q, queuosine. e, Schematic showing two mechanisms of polyamine depletion therapy. Only the combined treatment induces mild hallmarks of eIF5A hypusination deficiency and boosts the codon-specific translation defect induced by polyamine depletion. As described in Fig. 3, data in b–e are from the Th-MYCN mouse model. For all mean, n = 5. Panels a and e created in BioRender. Morscher, R. (2025) https://BioRender.com/75ofpvn (a); https://BioRender.com/iydko99 (e).
