scholarly journals EDF1 coordinates cellular responses to ribosome collisions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Niladri K Sinha ◽  
Alban Ordureau ◽  
Katharina Best ◽  
James A Saba ◽  
Boris Zinshteyn ◽  
...  
Keyword(s):  
Quality Control ◽  
Ribosomal Subunit ◽  
Transcriptional Response ◽  
Related Factor ◽  
Electron Microscopic ◽  
40S Ribosomal Subunit ◽  
Gradient Fractionation ◽  
Yeast Homolog ◽  
Early Transcriptional Response ◽  
Novel Protein

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.

scholarly journals yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1384 ◽  
Author(s):  
Kerstin Schmitt ◽  
Oliver Valerius
Keyword(s):  
Quality Control ◽  
Binding Proteins ◽  
Ribosomal Subunit ◽  
Stress Factors ◽  
Head Region ◽  
Ubiquitin E3 Ligase ◽  
Mrna Binding Proteins ◽  
40S Ribosomal Subunit ◽  
Mrna Binding

Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The Saccharomyces cerevisiae protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit (hr40S) and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the hr40S. We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of hr40S localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.

Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

1987 ◽  
Vol 45 ◽  
pp. 936-937
Author(s):  
Neng-Yu Zhang ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
Tom Obrig ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Uranyl Acetate ◽  
Reconstruction Method ◽  
Single Exposure ◽  
Electron Dose ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

scholarly journals Translation Initiation Factor eIF3b Contains a Nine-Bladed β-Propeller and Interacts with the 40S Ribosomal Subunit

Structure ◽  
2014 ◽  
Vol 22 (6) ◽  
pp. 923-930 ◽  
Author(s):  
Yi Liu ◽  
Piotr Neumann ◽  
Bernhard Kuhle ◽  
Thomas Monecke ◽  
Stephanie Schell ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
40S Ribosomal Subunit

scholarly journals Deciphering molecular details of the RAC–ribosome interaction by EPR spectroscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sandra J. Fries ◽  
Theresa S. Braun ◽  
Christoph Globisch ◽  
Christine Peter ◽  
Malte Drescher ◽  
...  
Keyword(s):  
Protein Folding ◽  
Epr Spectroscopy ◽  
De Novo ◽  
Contact Point ◽  
Ribosomal Subunit ◽  
Paramagnetic Resonance ◽  
40S Ribosomal Subunit ◽  
Site Directed Spin Labeling ◽  
Α Helix ◽  
Chaperone Complex

AbstractThe eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC–ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

2021 ◽  
Author(s):  
Haina Huang ◽  
Melissa Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

scholarly journals Stepwise maturation of the peptidyl transferase region of human mitoribosomes

2021 ◽  
Author(s):  
Tea Lenarcic ◽  
Mateusz Jaskolowski ◽  
Marc Leibundgut ◽  
Alain Scaiola ◽  
Tanja Schoenhut ◽  
...  
Keyword(s):  
Quality Control ◽  
16S Rrna ◽  
Active Site ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Ribosome Assembly ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by MRM2 methyltransferase and quality control interactions with a conserved mitochondrial GTPase MTG2 that contacts the sarcin ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

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