A hallmark of ribosomal RNA (rRNA) are 2'-O-methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum, many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2'-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.
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