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Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.
Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification
Ribosome biogenesis is essential for cellular function and involves rRNA synthesis, rRNA processing and modification, and ribosomal protein assembly. Ribosome biogenesis factors and small nucleolar RNA assist these events. Ribosomal maturation takes place in the nucleolus, the nucleoplasm, and the cytosol in a coordinated and controlled manner. For example, some ribosomal proteins are thought to be assembled in the cytoplasm based on the observations in Saccharomyces cerevisiae. Here, we used cellular fractionation to demonstrate that cleavage of the 20S intermediate, the precursor to mature 18S rRNA, does not occur in the nucleoplasm of Arabidopsis thaliana. It most likely occurs in the cytoplasm. Further, we verified the proposed localization of RPS10e, RPS26e, and RPL24a/b in the nucleus and RPP1 in the nucleolus of A. thaliana by ribosome profiling, immunofluorescence, and analysis of the localization of GFP fusion proteins. Our results suggest that the order of events during ribosomal protein assembly in the ribosome biogenesis pathway differs between plants and yeast.