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Ribosome assembly is an essential and carefully choreographed cellular process. In eukaryotes, several 100 proteins, distributed across the nucleolus, nucleus, and cytoplasm, co-ordinate the step-wise assembly of four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (RPs) into the mature ribosomal subunits. Due to the inherent complexity of the assembly process, functional studies identifying ribosome biogenesis factors and, more importantly, their precise functions and interplay are confined to a few and very well-established model organisms. Although best characterized in yeast (Saccharomyces cerevisiae), emerging links to disease and the discovery of additional layers of regulation have recently encouraged deeper analysis of the pathway in human cells. In archaea, ribosome biogenesis is less well-understood. However, their simpler sub-cellular structure should allow a less elaborated assembly procedure, potentially providing insights into the functional essentials of ribosome biogenesis that evolved long before the diversification of archaea and eukaryotes. Here, we use a comprehensive phylogenetic profiling setup, integrating targeted ortholog searches with automated scoring of protein domain architecture similarities and an assessment of when search sensitivity becomes limiting, to trace 301 curated eukaryotic ribosome biogenesis factors across 982 taxa spanning the tree of life and including 727 archaea. We show that both factor loss and lineage-specific modifications of factor function modulate ribosome biogenesis, and we highlight that limited sensitivity of the ortholog search can confound evolutionary conclusions. Projecting into the archaeal domain, we find that only few factors are consistently present across the analyzed taxa, and lineage-specific loss is common. While members of the Asgard group are not special with respect to their inventory of ribosome biogenesis factors (RBFs), they unite the highest number of orthologs to eukaryotic RBFs in one taxon. Using large ribosomal subunit maturation as an example, we demonstrate that archaea pursue a simplified version of the corresponding steps in eukaryotes. Much of the complexity of this process evolved on the eukaryotic lineage by the duplication of ribosomal proteins and their subsequent functional diversification into ribosome biogenesis factors. This highlights that studying ribosome biogenesis in archaea provides fundamental information also for understanding the process in eukaryotes.
Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.
Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.
The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Psi). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Psi1191 methylation. ESI MS analysis of acp-modified Psi-nucleosides in a DeltaNep1-mutant showed that Nep1 catalyzes the Psi1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a deltasnr35 mutant. This strongly suggests a dual Nep1 function, as Psi1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.
Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase
(2008)
In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA—U3, U14 and snR30—whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs. Keywords: ribosome biogenesis; RNA helicase; snoRNA