Fig 2: Riox1 and Riox2 expression in Hydra. a Alignment of both Hydra sequences, with highlighted iron-binding motif (HxD…H) (green) and predicted lysine-residue for 2OG binding (blue). b, c Expression of GFP-tagged HyRiox1 in Hydra animals displayed nuclear localisation and strong accumulation in nucleoli. d, e HyRiox2 is also localised in nuclei, but an accumulation in nucleoli is not detectable. DNA-stain: DAPI. Scalebar: 10 µm

Fig 3: Nuclear localisation of Riox1 and Riox2. The N-terminal extension domain of human RIOX1 has been shown to harbour the nuclear localisation signal (NLS) [28]. a Expression of GFP-tagged full-length RIOX1 in HeLa cells resulted mainly in nucleolar accumulation. b A RIOX1 deletion mutant lacking aa 1–31 localised to the cytoplasm. c Fusion of aa 1–45 of human RIOX1 resulted in nuclear GFP localisation with strong accumulation in the nucleoli. DNA stain: DAPI. Scalebar: 5 µm. d Predictions of NLS in Riox1 of other species with either NLSmapper (blue) or NLStradamus (pink) identified NLS in the N-terminal extension domains of Riox1, whereas for Riox2 no NLS were predicted

Fig 4: The ribosomal oxygenases (ROXs) are a subgroup of Fe(II) and 2OG-dependent oxygenases that modify the ribosome and are present in pro- and eukaryotes. a The human ROXs RIOX2/MINA53 and RIOX1/NO66 hydroxylate histidine residues in the ribosomal proteins RPL27A and RPL8, respectively, whereas (b) the E.coli ycfD protein hydroxlyates an arginine in Rpl16 [6]. Protein sequence and crystal structure analyses confirmed a similar protein-domain architecture for the three proteins [16]. c They consist of a JmjC-domain (red), a dimerization domain (brown) for homo-oligomerization and a winged-helix (WH) domain (blue). The aa triad HxD…H that coordinates the iron and is essential for catalytic activity is indicated in green (c)

Fig 5: Phylogenetic relationship of Riox1 and Riox2 JmjC domain sequences in Metazoa. Riox1- and Riox2-JmjC domain sequences from species used in this study were extracted from full-length protein sequences, aligned using ClustalW and maximum-likelihood analysis used for tree construction (IQ-TREE). The tree shown is a consensus tree with SH-like aLRT and ultrafast bootstrap (UFboot) values (numbers in parentheses SH-aLRT support (%)/ultrafast bootstrap support (%)) given as branch support values. Good branch support is confirmed with SH-aLRT > = 80% and UFboot > = 95%. The Tree is unrooted although the outgroup taxon ‘Trichoplax’ is drawn at root. The scale bar indicates 1.00 substitutions per site. The JmjC-domain of the E. coli ycfD ribosomal oxygenase (ecycfD) was included in the alignment to analyse its phylogenetic relationship to metazoan Riox1 and Riox2 proteins (indicated in red). Riox2-JmjC domain branch is highlighted with grey background shading to show its separate branch node relationship to Riox1-JmjC domains. Note, Riox2 is also present in Hydra vulgaris and Priapulus caudatus which both possess Riox1 and Riox2 orthologous genes as invertebrates. Species with a Riox1 gene, but which lack a Riox2 gene are highlighted in red