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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.
Genetic mutations underlying neurodegenerative disorders impair ribosomal DNA (rDNA) transcription suggesting that nucleolar dysfunction could be a novel pathomechanism in polyglutamine diseases and in certain forms of amyotrophic lateral sclerosis/frontotemporal dementia. Here, we investigated nucleolar activity in pre-symptomatic digenic models of Parkinson's disease (PD) that model the multifactorial aetiology of this disease. To this end, we analysed a novel mouse model mildly overexpressing mutant human α-synuclein (hA53T-SNCA) in a PTEN-induced kinase 1 (PINK1/PARK6) knockout background and mutant mice lacking both DJ-1 (also known as PARK7) and PINK1. We showed that overexpressed hA53T-SNCA localizes to the nucleolus. Moreover, these mutants show a progressive reduction of rDNA transcription linked to a reduced mouse lifespan. By contrast, rDNA transcription is preserved in DJ-1/PINK1 double knockout (DKO) mice. mRNA levels of the nucleolar transcription initiation factor 1A (TIF-IA, also known as RRN3) decrease in the substantia nigra of individuals with PD. Because loss of TIF-IA, as a tool to mimic nucleolar stress, increases oxidative stress and because DJ-1 and PINK1 mutations result in higher vulnerability to oxidative stress, we further explored the synergism between these PD-associated genes and impaired nucleolar function. By the conditional ablation of TIF-IA, we blocked ribosomal RNA (rRNA) synthesis in adult dopaminergic neurons in a DJ-1/PINK1 DKO background. However, the early phenotype of these triple knockout mice was similar to those mice exclusively lacking TIF-IA. These data sustain a model in which loss of DJ-1 and PINK1 does not impair nucleolar activity in a pre-symptomatic stage. This is the first study to analyse nucleolar function in digenic PD models. We can conclude that, at least in these models, the nucleolus is not as severely disrupted as previously shown in DA neurons from PD patients and neurotoxin-based PD mouse models. The results also show that the early increase in rDNA transcription and nucleolar integrity may represent specific homeostatic responses in these digenic pre-symptomatic PD models.
In this thesis the three dimensional solution strucutre of the RbfA protein from Thermotoga maritima was solved using multidimensional heteronuclear NMR spectroscopy. The RbfA protein binds to the helix I region of the 16S rRNA. To gain insights into the binding mode of RbfA to its target, a second RbfA construct from Helicobacter pylori was used. Comparison of the RbfA proteins with the published structure of RbfA from Escherichia coli, led to studies concerning the differences between proteins from thermophile and mesophile systems. In the second part of this thesis the native binding motive of the RbfA protein was identified. The RbfA protein binds to an alternate helix fold within the pre-sequence of the immature 16S rRNA.