Ribosome assembly factors Nsa2 and Rsa4 connect the ATPase Rea1 to the maturing catalytic center of the large subunit

Ribosome biogenesis is a highly complex process, which in eukaryotes depends on a myriad of assembly factors, including several energy-consuming enzymes. One of these is the ATPase Rea1, that is necessary for the formation of large ribosomal subunits. Rea1 is responsible for the removal of several assembly factors, including Rsa4, during a late step in 60S biogenesis. This release depends on a Rea1-generated pulling force, that is transmitted to Rsa4 and eventually results in its dissociation from pre-ribosomes. It is therefore of high interest to identify, which proteins or rRNA elements connect Rsa4 to the pre-ribosome, as these could transmit the Rea1 power stroke to the maturing 60S subunit and result in structural rearrangements at their binding site. This study builds on initial findings, that the 60S assembly factor Nsa2 is a putative interaction partner of Rsa4. Using genetic and biochemical approaches, I was able to verify the interaction and demonstrate, that it is essential for yeast growth. Furthermore, I was able to crystallize the Nsa2-Rsa4 hetero-dimer and the structure was solved in collaboration with the lab of Dr. Irmi Sinning (BZH, Heidelberg). A subsequent structurefunction analysis revealed the molecular details of the Nsa2-Rsa4 interaction and its impact on 60S biogenesis. Moreover, I was able to fit the Nsa2-Rsa4 crystal structure in an EM-volume of the Arx1 pre-ribosome, that places Nsa2 and Rsa4 at the nascent peptidyl transferase center (PTC). Here, Rsa4 is bound to the immature central protuberance and Nsa2 is oriented towards the nascent tRNA binding site. Using Nsa2 NMR structures, which were generated in collaboration with the lab of Dr. Elisar Barbar (Oregon State University), and crosslinking data from Dr. Sander Granneman (University of Edinburgh), I propose a model in which the globular C-domain of Nsa2 is located at the maturing peptidyl transferase center and the α-helical N-domain of Nsa2 reaches around immature rRNA helix 89 towards the P stalk region. Nsa2 and Rsa4 therefore connect Rea1 to the maturing PTC, which suggests an additional function of the Rea1-generated pulling force beyond the mere removal of assembly factors. The positioning and functional analysis of Nsa2 implies, that Rea1 exerts a mechanical force on immature helix 89, which is necessary for assembly of the catalytic center during 60S biogenesis

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

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center

NyarkoAfuaBiochemBiophyNetworkAssemblyFactors.pdf

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

J Cell Biol-2015-BaßlerJochenCorrection.pdf

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

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge