The Role of Energy Consuming Enzymes in 60S Ribosomal Subunit Assembly

Functional characterization of the Nug1 GTPase and the Rea1 ATPase and their role in 60S ribosome biogenesis

Isolierung eines nukleären 60S Prä-Ribosoms, das ein Exportintermediat ins Zytoplasma darstellt

Im Gegensatz zu anderen nukleozytoplasmatischen Transportprozessen in eukaryontischen Zellen, ist der Export von Ribosomen aus dem Zellkern ins Zytoplasma noch weitgehend unverstanden. In dieser Arbeit wird die Reinigung und Charakterisierung eines prä-ribosomalen 60S Partikels beschrieben, das ein spätes Exportintermediat aus dem Zellkern ins Zytoplasma darstellen könnte. Ausgehend von der ungewöhnlichen mtr2-33 Mutante (Mtr2p ist ein mRNA-Exportfaktor), die keinen mRNA-Exportdefekt, aber einen ribosomalen Export-defekt aufweist, wurden über genetische Ansätze die neuen Ribosomen-biogenesefaktoren Ecm1p, Nug1p und Nug2p identifiziert. Dabei sind Nug1p und Nug2p Mitglieder einer neuartigen evolutionär konservierten Proteinfamilie von GTPasen. Es konnte gezeigt werden, daß Nug1p und Nug2p mit 60S Prä-Ribosomen assoziieren. Entsprechend war es möglich, diese 60S prä-ribosomalen Partikel durch Affinitätsreinigung von Nug1p zu isolieren. Das Nug1p / Nug2p enthaltende Prä-Ribosom besteht aus den ribosomalen L-Proteinen, 27SB und 7S rRNA-Vorstufen und reifer 25S, 5.8S und 5S rRNA. Weiterhin enthält das 60S Prä-Ribosom mindestens 23 nichtribosomale Proteine, die überwiegend essentiell und evolutionär konserviert sind. Nachdem nug1 und nug2 ts Mutanten Defekte im 60S Export aufweisen, aber nicht in der rRNA-Prozessierung beeinträchtigt sind, schlage ich vor, daß das isolierte Nug1p-Partikel ein Intermediat während des Exportes der 60S Untereinheit aus dem Zellkern ins Zytoplasma darstellt

Interdependent action of KH domain proteins Krr1 and Dim2 drive the 40S platform assembly

Ribosome biogenesis begins in the nucleolus with the formation of 90S pre-ribosomes, from which pre-40S and pre-60S particles arise that subsequently follow separate maturation pathways. Here, we show how structurally related assembly factors, the KH domain proteins Krr1 and Dim2, participate in ribosome assembly. Initially, Dim2 (Pno1) orchestrates an early step in small subunit biogenesis through its binding to a distinct region of the 90S pre-ribosome. This involves Utp1 of the UTP-B module, and Utp14, an activator of the DEAH-box helicase Dhr1 that catalyzes the removal of U3 snoRNP from the 90S. Following this dismantling reaction, the pre-40S subunit emerges, but Dim2 relocates to the pre-40S platform domain, previously occupied in the 90S by the other KH factor Krr1 through its interaction with Rps14 and the UTP-C module. Our findings show how the structurally related Krr1 and Dim2 can control stepwise ribosome assembly during the 90S-to-pre-40S subunit transition

Mechanochemical Removal of Ribosome Biogenesis Factors from Nascent 60S Ribosomal Subunits

SummaryThe dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit

Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration

The ‘birth’ of the eukaryotic ribosome is preceded by RNA folding and processing reactions that depend on assembly factors and snoRNAs. The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5’>3’, but this directionality may not be accurate. Here, by analyzing the structure of series of novel 90S assembly intermediates isolated from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. This revealed that large parts of the 18S rRNA 3’ and central domains assemble first into the 90S, before the 5’ domain is stably integrated. This final incorporation depends on a physical contact between a heterotrimer Enp2-Bfr2-Lcp1 recruited to the flexible 5’ domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S pre-ribosome. Keeping the 5’ domain temporarily segregated from the 90S scaffold could provide an extra time to complete the multifaceted 5’ domain folding, which depends on a distinct set of snoRNAs and processing factors

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ~30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

Hierarchisierung von Risikofaktoren für schwere COVID-19-Erkrankungsverläufe im Kontext der COVID-19-Schutzimpfungen

Angesichts der derzeitigen Impfstoffknappheit geht mit den bundesweiten Schutzimpfungen gegen COVID-19 die Notwendigkeit einer Priorisierung bestimmter Bevölkerungsgruppen einher. Basierend auf den Empfehlungen der STIKO sollen zunächst Personen mit besonders hohem Risiko für schwere oder tödliche COVID-19-Verläufe oder beruflicher Exposition geimpft werden. Diese Empfehlungen stützen sich überwiegend auf internationale Studien - für den deutschen Versorgungskontext steht nur begrenzt Evidenz zur Bedeutung relevanter Risikofaktoren für einen schweren COVID-19-Verlauf zur Verfügung. Das Ziel der im Epidemiologischen Bulletin 19/2021 vorgestellten Studie war es, die Relevanz ausgewählter Vorerkrankungen für einen schweren COVID-19-Verlauf in der in Deutschland lebenden Bevölkerung empirisch zu überprüfen, Erkrankungen hinsichtlich ihres Risikos für einen schweren COVID-19-Verlauf zu ordnen und damit eine einfache, im Versorgungsalltag unkompliziert umsetzbare und dabei möglichst effektive Grundlage für die Impfrangfolge in der ambulanten ärztlichen Versorgung bilden

A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

The following corrections appear in the attached pdf labelled "Correction to Version of Record". The authors inadvertently omitted Woonghee Lee from the list of authors. The corrected author list and affiliations are noted. Revised acknowledgment paragraphs including Woonghee Lee’s funding source and contribution also appear in the correction. In addition, the authors noted the omission of details of NMR structure calculation from the Materials and Methods section. The relevant paragraph and the references associated with it are appended.Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation

A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation