Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

植物核糖体应激响应机制研究进展

核仁是真核细胞中重要的核结构,核糖体发生最初在核仁中进行,该过程涉及一系列复杂的反应,需要许多核仁相关因子参与。核糖体生物发生出现异常通常引起核仁结构紊乱,并导致细胞周期阻滞、细胞衰老甚至凋亡。核糖体应激响应机制在哺乳动物细胞中研究得较为深入,但在植物细胞中尚不明晰。尽管如此,人们逐渐发现某些植物特有的NAC转录因子家族成员在植物细胞中可能参与包括核糖体应激在内的多种胞内应激响应过程。此外,前期研究发现生长素系统与核糖体生物合成之间存在一种相互协调机制来调控植物发育。该文结合哺乳动物细胞中已知的核糖体应激响应通路,探讨植物细胞潜在的核糖体应激机制。     

Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis

Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors. Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis. Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis. Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes. Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA. Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes. Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.     

The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing

The ubiquitin-like SUMO system functions by a cyclic process of modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation-desumoylation cycles. For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMO-specific protease SENP3 is associated with nucleophosmin (NPM1), a crucial factor in ribosome biogenesis. SENP3 catalyses desumoylation of NPM1-SUMO2 conjugates in vitro and counteracts ARF-induced modification of NPM1 by SUMO2 in vivo. Intriguingly, depletion of SENP3 by short interfering RNA interferes with nucleolar ribosomal RNA processing and inhibits the conversion of the 32S rRNA species to the 28S form, thus phenocopying the processing defect observed on depletion of NPM1. Moreover, mimicking constitutive modification of NPM1 by SUMO2 interferes with 28S rRNA maturation. These results define SENP3 as an essential factor for ribosome biogenesis and suggest that deconjugation of SUMO2 from NPM1 by SENP3 is critically involved in 28S rRNA maturation.     

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in “half-mer” polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Δ cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Δ cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Δ cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.     

Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae

The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.     

The nucleolar protein SAHY1 is involved in pre-rRNA processing and normal plant growth

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A₃, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

SALT-HYPERSENSITIVE MUTANT 1, a nucleolar protein involved in ribosome biogenesis, regulates the auxin-mediated development of vegetative and reproductive tissues.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

The 60S associated ribosome biogenesis factor LSG1‐2 is required for 40S maturation in Arabidopsis thaliana

Ribosome biogenesis involves a large ensemble of trans‐acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre‐60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C‐terminus, which is highly charged in atLSG1‐2 but missing in atLSG1‐1. This C‐terminus of atLSG1‐2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1‐2 to the nucleolus. Furthermore, only atLSG1‐2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T‐DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1‐2 18S rRNA precursors accumulate and a 20S pre‐rRNA is detected, while the amount of pre‐rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre‐60S subunit maturation is important for the final steps of pre‐40S maturation in plants. In addition, the lsg1‐2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.     

Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C₂.     

Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.     

Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.