scholarly journals Structural basis for late maturation steps of the human mitoribosomal large subunit

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Miriam Cipullo ◽  
Genís Valentín Gesé ◽  
Anas Khawaja ◽  
B. Martin Hällberg ◽  
Joanna Rorbach
Keyword(s):  
16S Rrna ◽  
Large Subunit ◽  
Elongation Factor ◽  
Fine Tuning ◽  
Structural Basis ◽  
Critical Set ◽  
Mitochondrial Ribosomes ◽  
Assembly Factor ◽  
Peptidyl Transferase Centre ◽  
Late Maturation

AbstractMitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.

scholarly journals Structural basis for late maturation steps of the human mitoribosomal large subunit

2021 ◽  
Author(s):  
Joanna Rorbach ◽  
Miriam Cipullo ◽  
Genís Valentín Gesé ◽  
Anas Khawaja ◽  
Martin Hällberg
Keyword(s):  
16S Rrna ◽  
Large Subunit ◽  
Elongation Factor ◽  
Fine Tuning ◽  
Structural Basis ◽  
Critical Set ◽  
Mitochondrial Ribosomes ◽  
Assembly Factor ◽  
Peptidyl Transferase Centre ◽  
Late Maturation

Abstract Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2–6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our structures reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.

scholarly journals Structural basis for late maturation steps of the human mitoribosomal large subunit

2021 ◽  
Author(s):  
Joanna Rorbach ◽  
Miriam Cipullo ◽  
Genis Valentin Gese ◽  
Martin Hallberg ◽  
Anas Khawaja
Keyword(s):  
16S Rrna ◽  
Large Subunit ◽  
Elongation Factor ◽  
Fine Tuning ◽  
Structural Basis ◽  
Critical Set ◽  
Mitochondrial Ribosomes ◽  
Assembly Factor ◽  
Peptidyl Transferase Centre ◽  
Late Maturation

Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.

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.

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 Stepwise maturation of the peptidyl transferase region of human mitoribosomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tea Lenarčič ◽  
Mateusz Jaskolowski ◽  
Marc Leibundgut ◽  
Alain Scaiola ◽  
Tanja Schönhut ◽  
...  
Keyword(s):  
Quality Control ◽  
16S Rrna ◽  
Active Site ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Ribosome Assembly ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome

AbstractMitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the 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 the MRM2 methyltransferase and quality control interactions with the 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.

scholarly journals Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Demo ◽  
Howard B. Gamper ◽  
Anna B. Loveland ◽  
Isao Masuda ◽  
Christine E. Carbone ◽  
...  
Keyword(s):  
16S Rrna ◽  
Elongation Factor ◽  
Structural Basis ◽  
Elongation Factor G ◽  
Ribosomal Frameshifting ◽  
Elongation Cycle ◽  
70S Ribosome ◽  
Complex Features ◽  
A Site ◽  
Factor G

AbstractFrameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

scholarly journals Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

2020 ◽  
Vol 48 (14) ◽  
pp. 7924-7943 ◽  
Author(s):  
Priyanka Maiti ◽  
Hana Antonicka ◽  
Anne-Claude Gingras ◽  
Eric A Shoubridge ◽  
Antoni Barrientos
Keyword(s):  
Quality Control ◽  
16S Rrna ◽  
Large Subunit ◽  
Mitochondrial Protein ◽  
Small Gtpases ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Ribosomes ◽  
Family Protein ◽  
Assembly Factors ◽  
Subunit Joining

Abstract Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.

scholarly journals Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

2020 ◽  
Author(s):  
Gabriel Demo ◽  
Anna B Loveland ◽  
Egor Svidritskiy ◽  
Howard B Gamper ◽  
Ya-Ming Hou ◽  
...  
Keyword(s):  
16S Rrna ◽  
Elongation Factor ◽  
Structural Basis ◽  
Elongation Factor G ◽  
Ribosomal Frameshifting ◽  
Elongation Cycle ◽  
Complex Features ◽  
A Site ◽  
Factor G

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. Where and how in the elongation cycle +1-frameshifting occurs remains poorly understood. We captured six ~3.5-Å-resolution cryo-EM structures of ribosomal elongation complexes formed with the GTPase elongation factor G (EF-G). Three structures with a +1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G, the tRNA shifts to the +1-frame codon near the P site, whereas the freed mRNA base bulges between the P and E sites and stacks on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during mRNA translocation.

scholarly journals Multilocus phylogenies reveal three new truffle-like taxa and the traces of interspecific hybridization in Octaviania (Boletaceae, Boletales)

IMA Fungus ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takamichi Orihara ◽  
Rosanne Healy ◽  
Adriana Corrales ◽  
Matthew E. Smith
Keyword(s):  
Rna Polymerase Ii ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Gene Tree ◽  
Phylogenetic Network ◽  
Elongation Factor ◽  
Morphological Observation ◽  
Tree Comparison ◽  
Unknown Species ◽  
The Usa

ABSTRACTAmong many convergently evolved sequestrate fungal genera in Boletaceae (Boletales, Basidiomycota), the genus Octaviania is the most diverse. We recently collected many specimens of Octaviania subg. Octaviania, including several undescribed taxa, from Japan and the Americas. Here we describe two new species in subgenus Octaviania, O. tenuipes and O. tomentosa, from temperate to subtropical evergreen Fagaceae forests in Japan based on morphological observation and robust multilocus phylogenetic analyses (nrDNA ITS and partial large subunit [LSU], translation elongation factor 1-α gene [TEF1] and the largest subunit of RNA polymerase II gene [RPB1]). Based on specimens from the Americas as well as studies of the holotype, we also taxonomically re-evaluate O. asterosperma var. potteri. Our analysis suggests that O. asterosperma var. potteri is a distinct taxon within the subgenus Octaviania so we recognize this as O. potteri stat. nov. We unexpectedly collected O. potteri specimens from geographically widespread sites in the USA, Japan and Colombia. This is the first verified report of Octaviania from the South American continent. Our molecular analyses also revealed that the RPB1 sequence of one O. tenuipes specimen was identical to that of a closely related species, O. japonimontana, and that one O. potteri specimen from Minnesota had an RPB1 sequence of an unknown species of O. subg. Octaviania. Additionally, one O. japonimontana specimen had an unusually divergent TEF1 sequence. Gene-tree comparison and phylogenetic network analysis of the multilocus dataset suggest that these heterogenous sequences are most likely the result of previous inter- and intra-specific hybridization. We hypothesize that frequent hybridization events in Octaviania may have promoted the high genetic and species diversity found within the genus.

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