Michael Prattes, Irina Grishkovskaya, Victor-Valentin Hodirnau, Ingrid Rössler, Isabella Klein, Christina Hetzmannseder, Gertrude Zisser, Christian C Gruber, Karl Gruber, David Haselbach, Helmut Bergler
Nature Communications volume 12, Article number: 3483 (2021)
The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.
Lisa Kofler, Michael Prattes, Helmut Bergler
Int. J. Mol. Sci. 2020, 21(8), 2998
The synthesis of ribosomes is one of the central and most resource demanding processes in each living cell. As ribosome biogenesis is tightly linked with the regulation of the cell cycle, perturbation of ribosome formation can trigger severe diseases, including cancer. Eukaryotic ribosome biogenesis starts in the nucleolus with pre-rRNA transcription and the initial assembly steps, continues in the nucleoplasm and is finished in the cytoplasm. From start to end, this process is highly dynamic and finished within few minutes. Despite the tremendous progress made during the last decade, the coordination of the individual maturation steps is hard to unravel by a conventional methodology. In recent years small molecular compounds were identified that specifically block either rDNA transcription or distinct steps within the maturation pathway. As these inhibitors diffuse into the cell rapidly and block their target proteins within seconds, they represent excellent tools to investigate ribosome biogenesis. Here we review how the inhibitors affect ribosome biogenesis and discuss how these effects can be interpreted by taking the complex self-regulatory mechanisms of the pathway into account. With this we want to highlight the potential of low molecular weight inhibitors to approach the dynamic nature of the ribosome biogenesis pathway.
Michael Prattes, Yu-Hua Lo, Helmut Bergler, Robin E Stanley
Biomolecules 2019, 9(11), 715
AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.
Valentin Mitterer, Ramtin Shayan, Sébastien Ferreira-Cerca, Guillaume Murat, Tanja Enne, Dana Rinaldi, Sarah Weigl, Hajrija Omanic, Pierre-Emmanuel Gleizes, Dieter Kressler, Celia Plisson-Chastang, Brigitte Pertschy
Nat Commun 10, 2754 (2019)
Eukaryotic ribosomes are synthesized in a hierarchical process driven by a plethora of assembly factors, but how maturation events at physically distant sites on pre-ribosomes are coordinated is poorly understood. Using functional analyses and cryo-EM, we show that ribosomal protein Rps20 orchestrates communication between two multi-step maturation events across the pre-40S subunit. Our study reveals that during pre-40S maturation, formation of essential contacts between Rps20 and Rps3 permits assembly factor Ltv1 to recruit the Hrr25 kinase, thereby promoting Ltv1 phosphorylation. In parallel, a deeply buried Rps20 loop reaches to the opposite pre-40S side, where it stimulates Rio2 ATPase activity. Both cascades converge to the final maturation steps releasing Rio2 and phosphorylated Ltv1. We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.
Lisa Kofler, Michael Prattes and Helmut Bergler
Microbial Cell, Vol. 6, No. 10
The formation of new ribosomes is a fundamental cellular process for each living cell and is tightly interwoven with cell cycle control and proliferation. Minimal disturbances of this pathway can result in ribosomopathies including an increased risk for certain cancer types. Thus, targeting ribosome biogenesis is an emerging strategy in cancer therapy. However, due to its complex nature, we are only at the beginning to understand the dynamics of the ribosome biogenesis pathway. One arising approach that will help us to embrace the tight timely cascade of events that is needed to form a new ribosome is the use of targeted chemical inhibition. However, only very few specific chemical inhibitors of the ribosome biogenesis pathway have been identified so far. Here we review our recently published screen to identify novel inhibitors of the ribosome biogenesis pathway in yeast (Awad et al., 2019, BMC Biology). These inhibitors can provide novel tools for basic research and can serve as starting-points to develop new chemotherapeutics.
Ingrid Rössler, Julia Embacher, Benjamin Pillet, Guillaume Murat, Laura Liesinger, Jutta Hafner, Julia Judith Unterluggauer, Ruth Birner-Gruenberger, Dieter Kressler, Brigitte Pertschy
Nucleic Acids Research, Volume 47, Issue 13, 26 July 2019
Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.
Dominik Awad, Michael Prattes, Lisa Kofler, Ingrid Rössler, Mathias Loibl, Melanie Pertl, Gertrude Zisser, Heimo Wolinski, Brigitte Pertschy, Helmut Bergler
BMC Biology volume 17, Article number: 46 (2019)
Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential. Despite this large repertoire of potential targets, only very few chemical inhibitors of ribosome biogenesis are known so far. Such inhibitors are valuable tools to study this highly dynamic process and elucidate mechanistic details of individual maturation steps. Moreover, ribosome biogenesis is of particular importance for fast proliferating cells, suggesting its inhibition could be a valid strategy for treatment of tumors or infections. We systematically screened ~ 1000 substances for inhibitory effects on ribosome biogenesis using a microscopy-based screen scoring ribosomal subunit export defects. We identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both. Northern blot analysis demonstrates that these inhibitors cause a broad spectrum of different rRNA processing defects.
Gertrude Zisser, Uli Ohmayer, Christina Mauerhofer, Valentin Mitterer, Isabella Klein, Gerald N Rechberger, Heimo Wolinski, Michael Prattes, Brigitte Pertschy, Philipp Milkereit, Helmut Bergler
Nucleic Acids Research, Volume 46, Issue 6, 6 April 2018, Pages 3140–3151
The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation.
Michael Prattes, Mathias Loibl, Gertrude Zisser, Daniel Luschnig, Lisa Kappel, Ingrid Rössler, Manuela Grassegger, Altijana Hromic, Elmar Krieger, Karl Gruber, Brigitte Pertschy and Helmut Bergler.
Scientific Reports, 7:44751
The yeast ribosome biogenesis factor Drg1 and the multifunctional mammalian protein p97 are both members of the widespread enzyme family of AAA-ATPases. Although they act in different cellular pathways their enzymatic domains are still conserved. In this study we characterized mutant variants of Drg1 with amino acid exchanges at the interface between the D1 domain (one of two ATPase domains) and the regulatory N-terminal domain. The regulatory defects caused by these mutations resemble those of mutations in p97 that cause the hereditary syndrome IBMPFD. The finding that similar mutations in both proteins disturb regulation of ATPase activity was surprising based on the low sequence conservation of the N-domain. This indicates that these functionally diverged proteins still use similar mechanisms to regulate their enzymatic activity.
Benjamin Pillet, Valentin Mitterer, Dieter Kressler, and Brigitte Pertschy
Bioessays, 2017, 39(1):1-12
In this review, we summarize the current knowledge of a novel class of proteins, specific chaperones for individual ribosomal proteins. Please see also the nice editorial of the issue on this topic:
Valentin Mitterer, Nadine Gantenbein, Ruth Birner-Gruenberger, Guillaume Murat, Helmut Bergler, Dieter Kressler, and Brigitte Pertschy.
Scientific Reports 2016, 6:36714
In our previous study we showed that Rps3 forms dimers, and that Yar1 is bound to only one of these two Rps3 molecules in vivo. Here, we investigated the nuclear import of this intriguing complex. Interestingly, the nuclear localization sequence of Rps3 is directly adjacent to the Yar1 binding site in the very N-terminal part of Rps3, and Yar1 competes with importins for Rps3 binding. Still, we found complexes containing Rps3, Yar1 and importins in vivo, suggesting that Rps3 can be imported in a complex containing importin bound to one Rps3 molecule and Yar1 to the second Rps3 in the dimer.
Valentin Mitterer, Guillaume Murat, Stéphane Réty, Magali Blaud, Lila Delbos, Tamsyn Stanborough, Helmut Bergler, Nicolas Leulliot, Dieter Kressler and Brigitte Pertschy.
Nature Communications 2016, 7:10336
In this study we report the intriguing stepwise assembly mechanism of ribosomal protein S3.S3 is imported into the nucleus in a dimeric conformation with it's N-domain rotated relative to the C-domain and bound to the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3.
Tamsyn Stanborough, Johannes Niederhauser, Barbara Koch, Helmut Bergler and Brigitte Pertschy.
FEBS Letters 2014, 588(5):569-64
We previously showed that yeast Rps3 interacts with the ankyrin repeat protein Yar1 in the course of the ribosome biogenesis pathway (see below, Koch et al.2012). Here, we made the exciting discovery that the human Rps3 protein also interacts with an ankyrin repeat protein, the NF-κB inhibitor IκBα. We suggest that, while the interaction of yeast Rps3 with ankyrin repeat proteins ensures that enough Rps3 is provided to the ribosome biogenesis pathway, the interaction of human Rps3 with IκBα ensures maintenance of an Rps3 pool for the NF-κB pathway.
Mathias Loibl, Isabella Klein, Michael Prattes, Claudia Schmidt, Lisa Kappel, Gertrude Zisser, Anna Gungl, Elmar Krieger, Brigitte Pertschy and Helmut Bergler
The Journal of Biological Chemistry 2014, 289(7):3913-3922
In this study we characterized the mechanism of action of diazaborine, which is until now the only known inhibitor of eukaryotic ribosome biogenesis. We discovered that diazaborine specifically binds to the AAA-ATPase Drg1 and inhibits ATP hydrolysis. This blocks release of Rlp24 from pre-60S particles by Drg1 (see also below, Kappel et al. 2012), thereby inhibiting the progression of ribosome biogenesis.
Lisa Kappel, Mathias Loibl, Gertrude Zisser, Isabella Klein, Gernot Fruhmann, Christof Gruber, Stefan Unterweger, Gerald Rechberger, Brigitte Pertschy and Helmut Bergler.
The Journal of Cell Biology 2012, 199(5):771-782
The pre-60S component Rlp24 accompanies 60S precursors from the nucleus into the cytoplasm. In this study, we discovered that in the cytoplasm, Rlp24 is bound by the AAA-ATPase Drg1. This interaction stimulates the ATPase activity of Drg1, which is utilized to release Rlp24 from the particle. This release is a key event in large subunit formation that is a prerequisite for further progression of cytoplasmic pre-60S maturation.
Jochen Baßler, Isabella Klein, Claudia Schmidt, Martina Kallas, Emma Thomson, Maria Anna Wagner, Bettina Bradatsch, Gerald Rechberger, Heimo Strohmaier, Ed Hurt and Helmut Bergler.
Molecular and Cellular Biology 2012, 32(24):4898-4912
Ribosome precursors are assembled in the nucleus and after a series of maturation steps, they are transported through the nuclear pores into the cytoplasm, where they undergo final maturation. In this study, we discovered, that Bud20 is a shuttling factor that accompanies pre-60S particles through the nuclear pore and is necessary for efficient export of the particles. We suggest that the function of Bud20 is to facilitate pre-60S export.
Barbara Koch, Valentin Mitterer, Johannes Niederhauser, Tamsyn Stanborough, Guillaume Murat, Gerald Rechberger, Helmut Bergler, Dieter Kressler and Brigitte Pertschy.
Journal of Biological Chemistry 2012, 287(26):21806-21815
Ribosomal proteins are synthesized in the cytoplasm, but most of them assemble with pre-ribosomal particles in the nucleus. Here we discovered that the ankyrin repeat protein Yar1 binds to the ribosomal protein Rps3 and acts as a specific chaperone for Rps3 that ensures its solubility on the path to its assembly site. Our results open the possibility that protection of ribosomal proteins by specific chaperones might be a general mechanism that ensures optimal rates of ribosome biogenesis.
Dieter Kressler, Ed Hurt, Helmut Bergler and Jochen Baßler.
Biochimica et Biophysica Acta 2012, 1823(1):92-100
In this review, we summarize the current knowledge on Rea1, Rix7 and Drg1, AAA-ATPases which participate in ribosome biogenesis. All three proteins function in the release of non-ribosomal proteins from pre-ribosomal particles. We propose the model that in the course of substrate release, the force generated upon ATP hydrolysis triggers major transitions within pre-60S particles, which are essential for downstream maturation processes.