scholarly journals Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

2019 ◽  
Author(s):  
Florent Waltz ◽  
Heddy Soufari ◽  
Anthony Bochler ◽  
Philippe Giegé ◽  
Yaser Hashem
Keyword(s):  
Ribosomal Proteins ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Pentatricopeptide Repeat ◽  
Structural Investigations ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Eukaryotic Species ◽  
Specific Proteins ◽  
Pentatricopeptide Repeat Proteins

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6–10. In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

scholarly journals Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

2020 ◽  
Vol 117 (47) ◽  
pp. 29851-29861 ◽  
Author(s):  
Heddy Soufari ◽  
Florent Waltz ◽  
Camila Parrot ◽  
Stéphanie Durrieu-Gaillard ◽  
Anthony Bochler ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Large Subunit ◽  
Microscopy Study ◽  
Small Subunit ◽  
Electron Microscopy Study ◽  
Mitochondrial Ribosomes ◽  
Cryo Electron Microscopy ◽  
Parasite Survival ◽  
Assembly Intermediate ◽  
Early Translation

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

scholarly journals Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

2020 ◽  
Author(s):  
Heddy Soufari ◽  
Florent Waltz ◽  
Camila Parrot ◽  
Stéphanie Durrieu ◽  
Anthony Bochler ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Large Subunit ◽  
Microscopy Study ◽  
Small Subunit ◽  
Electron Microscopy Study ◽  
Mitochondrial Ribosomes ◽  
Cryo Electron Microscopy ◽  
Parasite Survival ◽  
Assembly Intermediate ◽  
Early Translation

AbstractKinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis1. They possess a single large mitochondrion, essential for the parasite survival2. In kinetoplastids mitochondrion, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes3. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes4,5. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit3,6. However, several important aspects mainly related to the large subunit remain elusive, such as the structure and maturation of its ribosomal RNA3. Here, we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the large subunit as well as previously unidentified ribosomal proteins. Most importantly, we introduce the structure of an LSU assembly intermediate in presence of 16 identified maturation factors. These maturation factors act both on the intersubunit and solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

scholarly journals Pentatricopeptide Repeat Proteins in Trypanosoma brucei Function in Mitochondrial Ribosomes

2007 ◽  
Vol 27 (19) ◽  
pp. 6876-6888 ◽  
Author(s):  
Mascha Pusnik ◽  
Ian Small ◽  
Laurie K. Read ◽  
Thomas Fabbro ◽  
André Schneider
Keyword(s):  
Trypanosoma Brucei ◽  
Large Subunit ◽  
Direct Interaction ◽  
Pentatricopeptide Repeat ◽  
Amino Acid Motif ◽  
Mitochondrial Ribosomes ◽  
Repeat Proteins ◽  
Ppr Proteins ◽  
Pentatricopeptide Repeat Proteins ◽  
Large Subunit Rrna

ABSTRACT The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A comparative analysis shows that seven out of eight selected PPR proteins are mitochondrially localized and essential for oxidative phosphorylation. Six of these are required for the stabilization of mitochondrial rRNAs and, like ribosomes, are associated with the mitochondrial membranes. Furthermore, one of the PPR proteins copurifies with the large subunit rRNA. Finally, ablation of all of the PPR proteins that were tested induces degradation of the other PPR proteins, indicating that they function in concert. Our results show that a significant number of trypanosomal PPR proteins are individually essential for the maintenance and/or biogenesis of mitochondrial rRNAs.

scholarly journals Abnormal Expression of Mitochondrial Ribosomal Proteins and Their Encoding Genes with Cell Apoptosis and Diseases

2020 ◽  
Vol 21 (22) ◽  
pp. 8879
Author(s):  
Guomin Huang ◽  
Hongyan Li ◽  
Hong Zhang
Keyword(s):  
Cell Apoptosis ◽  
Ribosomal Proteins ◽  
Mitochondrial Respiratory Chain ◽  
Basic Structure ◽  
Disease Diagnosis ◽  
Mitochondrial Ribosomes ◽  
Abnormal Expression ◽  
Metabolism Disorder ◽  
Mitochondrial Ribosome ◽  
Inducing Factors

Mammalian mitochondrial ribosomes translate 13 proteins encoded by mitochondrial genes, all of which play roles in the mitochondrial respiratory chain. After a long period of reconstruction, mitochondrial ribosomes are the most protein-rich ribosomes. Mitochondrial ribosomal proteins (MRPs) are encoded by nuclear genes, synthesized in the cytoplasm and then, transported to the mitochondria to be assembled into mitochondrial ribosomes. MRPs not only play a role in mitochondrial oxidative phosphorylation (OXPHOS). Moreover, they participate in the regulation of cell state as apoptosis inducing factors. Abnormal expressions of MRPs will lead to mitochondrial metabolism disorder, cell dysfunction, etc. Many researches have demonstrated the abnormal expression of MRPs in various tumors. This paper reviews the basic structure of mitochondrial ribosome, focuses on the structure and function of MRPs, and their relationships with cell apoptosis and diseases. It provides a reference for the study of the function of MRPs and the disease diagnosis and treatment.

scholarly journals The Chlamydomonas mitochondrial ribosome: how to build a ribosome from RNA fragments

2021 ◽  
Author(s):  
Florent WALTZ ◽  
Thalia Salinas-Giegé ◽  
Robert Englmeier ◽  
Herrade Meichel ◽  
Heddy Soufari ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Electron Tomography ◽  
Functional Study ◽  
Messenger Rnas ◽  
Novel Proteins ◽  
Contact Points ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Ribosome ◽  
Membrane Tethering ◽  
Specific Proteins

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Novel proteins, mainly helical repeat proteins, including OPR, PPR and mTERF proteins are found in Chlamydomonas mitoribosome, revealing the first structure of an OPR protein in complex with its RNA target. Targeted amiRNA silencing indicated that the novel ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of the mitoribosome diversity and the various strategies they adopt for membrane tethering.

scholarly journals The yeast protein Mam33 functions in the assembly of the mitochondrial ribosome

2019 ◽  
Vol 294 (25) ◽  
pp. 9813-9829 ◽  
Author(s):  
Gabrielle A. Hillman ◽  
Michael F. Henry
Keyword(s):  
Ribosomal Proteins ◽  
Matrix Protein ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Binding Assays ◽  
Safe Delivery ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Evolutionarily Conserved

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. Although remarkable progress has been made toward understanding the structure of mitoribosomes, the pathways and factors that facilitate their biogenesis remain largely unknown. The long unstructured domains of unassembled ribosomal proteins are highly prone to misfolding and often require dedicated chaperones to prevent aggregation. To date, chaperones that ensure safe delivery to the assembling ribosome have not been identified in the mitochondrion. In this study, a respiratory synthetic lethality screen revealed a role for an evolutionarily conserved mitochondrial matrix protein called Mam33 in Saccharomyces cerevisiae mitoribosome biogenesis. We found that the absence of Mam33 results in misassembled, aggregated ribosomes and a respiratory lethal phenotype in combination with other ribosome-assembly mutants. Using sucrose gradient sedimentation, native affinity purifications, in vitro binding assays, and SILAC-based quantitative proteomics, we found that Mam33 does not associate with the mature mitoribosome, but directly binds a subset of unassembled large subunit proteins. Based on these data, we propose that Mam33 binds specific mitoribosomal proteins to ensure proper assembly.

scholarly journals Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins

2021 ◽  
Vol 22 (11) ◽  
pp. 5496
Author(s):  
Annalisa Pecoraro ◽  
Martina Pagano ◽  
Giulia Russo ◽  
Annapina Russo
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Protein Biosynthesis ◽  
Cellular Respiration ◽  
Mitochondrial Ribosomes ◽  
Ribonucleoprotein Complexes ◽  
Mitochondrial Ribosome ◽  
Energy Consuming ◽  
Cell Growth And Proliferation

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

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