Abstract
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.
Highlights
On, the peptidyltransferase reaction of the ribosome was shown to be resistant to protein degradative treatment[1]
The number of 17 box C/D small nucleolar RNAs (snoRNAs) (Fig. 1) identified in D. discoideum prior to this study is relatively small for normally-sized ribosomal RNA (rRNA) sequences[42] compared to orthologous RNAs found in other species[62]
We employed an in silicoapproach for the identification of novel box C/D snoRNAs by using the probabilistic model-dependent search tool snoScan[48], which we combined with RNA-seq analyses
Summary
The peptidyltransferase reaction of the ribosome was shown to be resistant to protein degradative treatment[1]. The most prominent nucleotide modifications in rRNA are 2′-O-ribose methylation (2′-O-Me) and pseudouridylation (Ψ) that are introduced site- These modifications are thought to be important for RNA folding, ribosome stability and translational fidelity[7,8,9]. Ribose methylations and pseudouridylations in eukaryotes are introduced in rRNA site- by small nucleolar ribonucleoprotein particles (snoRNPs), as summarized r ecently[6,10,16]. D. discoideum single cells usually propagate by mitotic division; upon starvation, a complex developmental process is initiated, in which about 100,000 cells aggregate to form a multicellular mobile slug after 16 h, resulting in a fruiting body within 24 h38. To these, sequencing of cloned fragments yielded 17 box C/D and one box H/ACA snoRNA(s) in D. discoideum, besides other ncRNAs
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