Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism

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

Analyse structurale de la biogenèse de la petite sous-unité ribosomique eucaryote par cryo-microscopie électronique et analyse d'images

Ribosome assembly is a complex process that requires the intervention of more than 200 assembly factors (AFs). These proteins are essential for the processing and modification of ribosomal RNAs, as well as the structural assembly of ribosomal subunits. This mechanism, highly studied in yeast, generally conserved in eukaryotes, but has become more complex with evolution in higher eukaryotes. In addition, defects in ribosome synthesis have recently been associated with a list of human genetic diseases (called ribosomopathies) and cancers, via ribosome biogenesis disease. Numerous molecular and functional studies then made it possible to define several successive stages of cytoplasmic maturation of pre-40S particles in human and yeast. It is now crucial to incorporate these highly detailed molecular descriptions of ribosome maturation events into a three-dimensional view of ribosome assembly and to understand the structural remodeling of pre-ribosomal maturation particles. Using tandem purification methods, coupled with cryo-electron microscopy and isolated particle analysis, I have determined several high-resolution 3D structures of cytoplasmic pre-40S particles, in yeast and human, at different maturation steps. First, i determined the 3D structure of the pre-40S particles, purified using AF Tsr1-FPZ as a bait at 3.1 Å resolution. Structural heterogeneity tests indicated that the beak and platform domains are dynamic zones, and sheds new light on the structural remodeling events occurring during 40S subunit assembly. Moreover, in collaboration with the team of Dr. Brigitte Pertschy, we have determined the 3D structure of yeast cytoplasmic pre-40S particles carrying point mutations on Rps20. Our atomic models have allowed to highlight a close relationship between the correct assembly of Rps20 and the release of AFs Ltv1 and Rio2 from the maturing small ribosomal subunit. Finally, I also determined the 3D structures of human pre-40S particles trapped at a very late cytoplasmic maturation step, with a resolution of ~3 Å. This work was performed in collaboration with Prof. Ulrike Kutay's team (ETH Zurich). These data allowed us to uncover new steps in the cytoplasmic maturation of human pre-40S particles. This structural study allows us to propose new molecular mechanisms underlying the final steps of eukaryotic ribosomal assembly.L'assemblage des deux sous-unités ribosomiques (appelées 40S et 60S chez les eucaryotes) est un processus complexe, qui nécessite l'intervention de plus de 200 co-facteurs de maturation (CFM). Pour former des sous-unités ribosomiques matures et fonctionnelles, les CFM sont nécessaires à la maturation des ARN ribosomiques (ARNr), mais également à leur repliement correct et à leur assemblage avec les protéines ribosomiques (Rps). Malgré une identification quasi-exhaustive de ces CFM chez la levure et l'humain, leur fonction moléculaire précise reste à élucider. Par ailleurs, des défauts dans la synthèse des ribosomes ont récemment été associés à une liste croissante de maladies génétiques humaines (appelées ribosomopathies) et de cancers, ce qui nécessite une compréhension précise de chaque étape des mécanismes de biogenèse des ribosomes. De nombreuses études moléculaires et fonctionnelles ont récemment permis de définir plusieurs étapes successives de maturation cytoplasmique des particules pré-40S eucaryotes. Il est maintenant crucial d'intégrer ces descriptions moléculaires très détaillées des événements de maturation des ribosomes dans une vision tridimensionnelle de l'assemblage des ribosomes, et de comprendre le remodelage structural que les particules pré-ribosomiques peuvent subir au cours de leur maturation. Dans ce but j'ai déterminé, par cryo-microscopie électronique en transmission et analyse d'images, la structure 3D de précurseurs de la petite sous-unité ribosomique, purifiés à différentes étapes de maturation, chez l'Homme et la levure. Dans un premier temps, j'ai utilisé le CFM Tsr1 comme appât de purification dans des levures de phénotype sauvage, et déterminé la structure haute résolution de ces particules pré-40S cytoplasmiques. Ceci m'a permis de mettre en évidence trois étapes de maturations successives, commençant par l'autophosphorylation et le relargage du CFM Ltv1 et aboutissant à la flexibilité de la plateforme de la particule pré-40S. Ensuite, en collaboration avec le Dr Brigitte Pertschy (Graz, Autriche) j'ai déterminé la structure de particules pré-40S cytoplasmiques de levure, portant une mutation sur la protéine Rps20.Ceci a permis de mieux caractériser le rôle de Rps20 dans le relargage des CFM Rio2 et Ltv1 lors de l'assemblage de la sous-unité 40S. Enfin, j'ai déterminé la structure 3D des particules pré-40S humaines purifiées à un stade cytoplasmique tardif, à une résolution de 3 Å. Ce travail a été mené en collaboration avec l'équipe du Pr. Ulrike Kutay (ETH Zurich). Cette analyse nous a permis de localiser pour la première fois le CFM RIO1 sur les particules pré-40S cytoplasmiques tardives. En outre, nos résultats nous ont permis de mettre en lumière l'existence de remodelages structuraux inédits dans les dernières étapes de la maturation de la petite sous-unité ribosomique humaine

Structural analysis of the eukaryotic small ribosomal subunit biogenesis by cryo-electron microscopy and image analysis

L'assemblage des deux sous-unités ribosomiques (appelées 40S et 60S chez les eucaryotes) est un processus complexe, qui nécessite l'intervention de plus de 200 co-facteurs de maturation (CFM). Pour former des sous-unités ribosomiques matures et fonctionnelles, les CFM sont nécessaires à la maturation des ARN ribosomiques (ARNr), mais également à leur repliement correct et à leur assemblage avec les protéines ribosomiques (Rps). Malgré une identification quasi-exhaustive de ces CFM chez la levure et l'humain, leur fonction moléculaire précise reste à élucider. Par ailleurs, des défauts dans la synthèse des ribosomes ont récemment été associés à une liste croissante de maladies génétiques humaines (appelées ribosomopathies) et de cancers, ce qui nécessite une compréhension précise de chaque étape des mécanismes de biogenèse des ribosomes. De nombreuses études moléculaires et fonctionnelles ont récemment permis de définir plusieurs étapes successives de maturation cytoplasmique des particules pré-40S eucaryotes. Il est maintenant crucial d'intégrer ces descriptions moléculaires très détaillées des événements de maturation des ribosomes dans une vision tridimensionnelle de l'assemblage des ribosomes, et de comprendre le remodelage structural que les particules pré-ribosomiques peuvent subir au cours de leur maturation. Dans ce but j'ai déterminé, par cryo-microscopie électronique en transmission et analyse d'images, la structure 3D de précurseurs de la petite sous-unité ribosomique, purifiés à différentes étapes de maturation, chez l'Homme et la levure. Dans un premier temps, j'ai utilisé le CFM Tsr1 comme appât de purification dans des levures de phénotype sauvage, et déterminé la structure haute résolution de ces particules pré-40S cytoplasmiques. Ceci m'a permis de mettre en évidence trois étapes de maturations successives, commençant par l'autophosphorylation et le relargage du CFM Ltv1 et aboutissant à la flexibilité de la plateforme de la particule pré-40S. Ensuite, en collaboration avec le Dr Brigitte Pertschy (Graz, Autriche) j'ai déterminé la structure de particules pré-40S cytoplasmiques de levure, portant une mutation sur la protéine Rps20.Ceci a permis de mieux caractériser le rôle de Rps20 dans le relargage des CFM Rio2 et Ltv1 lors de l'assemblage de la sous-unité 40S. Enfin, j'ai déterminé la structure 3D des particules pré-40S humaines purifiées à un stade cytoplasmique tardif, à une résolution de 3 Å. Ce travail a été mené en collaboration avec l'équipe du Pr. Ulrike Kutay (ETH Zurich). Cette analyse nous a permis de localiser pour la première fois le CFM RIO1 sur les particules pré-40S cytoplasmiques tardives. En outre, nos résultats nous ont permis de mettre en lumière l'existence de remodelages structuraux inédits dans les dernières étapes de la maturation de la petite sous-unité ribosomique humaine.Ribosome assembly is a complex process that requires the intervention of more than 200 assembly factors (AFs). These proteins are essential for the processing and modification of ribosomal RNAs, as well as the structural assembly of ribosomal subunits. This mechanism, highly studied in yeast, generally conserved in eukaryotes, but has become more complex with evolution in higher eukaryotes. In addition, defects in ribosome synthesis have recently been associated with a list of human genetic diseases (called ribosomopathies) and cancers, via ribosome biogenesis disease. Numerous molecular and functional studies then made it possible to define several successive stages of cytoplasmic maturation of pre-40S particles in human and yeast. It is now crucial to incorporate these highly detailed molecular descriptions of ribosome maturation events into a three-dimensional view of ribosome assembly and to understand the structural remodeling of pre-ribosomal maturation particles. Using tandem purification methods, coupled with cryo-electron microscopy and isolated particle analysis, I have determined several high-resolution 3D structures of cytoplasmic pre-40S particles, in yeast and human, at different maturation steps. First, i determined the 3D structure of the pre-40S particles, purified using AF Tsr1-FPZ as a bait at 3.1 Å resolution. Structural heterogeneity tests indicated that the beak and platform domains are dynamic zones, and sheds new light on the structural remodeling events occurring during 40S subunit assembly. Moreover, in collaboration with the team of Dr. Brigitte Pertschy, we have determined the 3D structure of yeast cytoplasmic pre-40S particles carrying point mutations on Rps20. Our atomic models have allowed to highlight a close relationship between the correct assembly of Rps20 and the release of AFs Ltv1 and Rio2 from the maturing small ribosomal subunit. Finally, I also determined the 3D structures of human pre-40S particles trapped at a very late cytoplasmic maturation step, with a resolution of ~3 Å. This work was performed in collaboration with Prof. Ulrike Kutay's team (ETH Zurich). These data allowed us to uncover new steps in the cytoplasmic maturation of human pre-40S particles. This structural study allows us to propose new molecular mechanisms underlying the final steps of eukaryotic ribosomal assembly

The Rio1p ATPase hinders premature entry into translation of late pre-40S pre-ribosomal particles

Cytoplasmic maturation of precursors to the small ribosomal subunit in yeast requires the intervention of a dozen assembly factors (AFs), the precise roles of which remain elusive. One of these is Rio1p that seems to intervene at a late step of pre-40S particle maturation. We have investigated the role played by Rio1p in the dynamic association and dissociation of AFs with and from pre-40S particles. Our results indicate that Rio1p depletion leads to the stalling of at least 4 AFs (Nob1p, Tsr1p, Pno1p/Dim2p and Fap7p) in 80S-like particles. We conclude that Rio1p is important for the timely release of these factors from 80S-like particles. In addition, we present immunoprecipitation and electron microscopy evidence suggesting that when Rio1p is depleted, a subset of Nob1p-containing pre-40S particles associate with translating polysomes. Using Nob1p as bait, we purified pre-40S particles from cells lacking Rio1p and performed ribosome profiling experiments which suggest that immature 40S subunits can carry out translation elongation. We conclude that lack of Rio1p allows premature entry of pre-40S particles in the translation process and that the presence of Nob1p and of the 18S rRNA 3 extension in the 20S pre-rRNA is not incompatible with translation elongation

The Rio1p ATPase hinders premature entry into translation of late pre-40S pre-ribosomal particles

International audienc

Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits

Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021

Background: Diabetes is one of the leading causes of death and disability worldwide, and affects people regardless of country, age group, or sex. Using the most recent evidentiary and analytical framework from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD), we produced location-specific, age-specific, and sex-specific estimates of diabetes prevalence and burden from 1990 to 2021, the proportion of type 1 and type 2 diabetes in 2021, the proportion of the type 2 diabetes burden attributable to selected risk factors, and projections of diabetes prevalence through 2050. Methods: Estimates of diabetes prevalence and burden were computed in 204 countries and territories, across 25 age groups, for males and females separately and combined; these estimates comprised lost years of healthy life, measured in disability-adjusted life-years (DALYs; defined as the sum of years of life lost [YLLs] and years lived with disability [YLDs]). We used the Cause of Death Ensemble model (CODEm) approach to estimate deaths due to diabetes, incorporating 25 666 location-years of data from vital registration and verbal autopsy reports in separate total (including both type 1 and type 2 diabetes) and type-specific models. Other forms of diabetes, including gestational and monogenic diabetes, were not explicitly modelled. Total and type 1 diabetes prevalence was estimated by use of a Bayesian meta-regression modelling tool, DisMod-MR 2.1, to analyse 1527 location-years of data from the scientific literature, survey microdata, and insurance claims; type 2 diabetes estimates were computed by subtracting type 1 diabetes from total estimates. Mortality and prevalence estimates, along with standard life expectancy and disability weights, were used to calculate YLLs, YLDs, and DALYs. When appropriate, we extrapolated estimates to a hypothetical population with a standardised age structure to allow comparison in populations with different age structures. We used the comparative risk assessment framework to estimate the risk-attributable type 2 diabetes burden for 16 risk factors falling under risk categories including environmental and occupational factors, tobacco use, high alcohol use, high body-mass index (BMI), dietary factors, and low physical activity. Using a regression framework, we forecast type 1 and type 2 diabetes prevalence through 2050 with Socio-demographic Index (SDI) and high BMI as predictors, respectively. Findings: In 2021, there were 529 million (95% uncertainty interval [UI] 500-564) people living with diabetes worldwide, and the global age-standardised total diabetes prevalence was 6·1% (5·8-6·5). At the super-region level, the highest age-standardised rates were observed in north Africa and the Middle East (9·3% [8·7-9·9]) and, at the regional level, in Oceania (12·3% [11·5-13·0]). Nationally, Qatar had the world's highest age-specific prevalence of diabetes, at 76·1% (73·1-79·5) in individuals aged 75-79 years. Total diabetes prevalence-especially among older adults-primarily reflects type 2 diabetes, which in 2021 accounted for 96·0% (95·1-96·8) of diabetes cases and 95·4% (94·9-95·9) of diabetes DALYs worldwide. In 2021, 52·2% (25·5-71·8) of global type 2 diabetes DALYs were attributable to high BMI. The contribution of high BMI to type 2 diabetes DALYs rose by 24·3% (18·5-30·4) worldwide between 1990 and 2021. By 2050, more than 1·31 billion (1·22-1·39) people are projected to have diabetes, with expected age-standardised total diabetes prevalence rates greater than 10% in two super-regions: 16·8% (16·1-17·6) in north Africa and the Middle East and 11·3% (10·8-11·9) in Latin America and Caribbean. By 2050, 89 (43·6%) of 204 countries and territories will have an age-standardised rate greater than 10%. Interpretation: Diabetes remains a substantial public health issue. Type 2 diabetes, which makes up the bulk of diabetes cases, is largely preventable and, in some cases, potentially reversible if identified and managed early in the disease course. However, all evidence indicates that diabetes prevalence is increasing worldwide, primarily due to a rise in obesity caused by multiple factors. Preventing and controlling type 2 diabetes remains an ongoing challenge. It is essential to better understand disparities in risk factor profiles and diabetes burden across populations, to inform strategies to successfully control diabetes risk factors within the context of multiple and complex drivers. Funding: Bill & Melinda Gates Foundation