scholarly journals Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

2020 ◽  
Vol 48 (22) ◽  
pp. 12929-12942 ◽  
Author(s):  
Elena Lavdovskaia ◽  
Kärt Denks ◽  
Franziska Nadler ◽  
Emely Steube ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Dual Function ◽  
Large Ribosomal Subunit ◽  
Ribosome Recycling Factor ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Assembly Intermediate

Abstract Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

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 Xenopus LSm Proteins Bind U8 snoRNA via an Internal Evolutionarily Conserved Octamer Sequence

2002 ◽  
Vol 22 (12) ◽  
pp. 4101-4112 ◽  
Author(s):  
Nenad Tomasevic ◽  
Brenda A. Peculis
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
High Specificity ◽  
Sm Proteins ◽  
U6 Snrna ◽  
Evolutionarily Conserved

ABSTRACT U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.

scholarly journals Structure and dynamics of bacterial ribosome biogenesis

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160181 ◽  
Author(s):  
Joseph H. Davis ◽  
James R. Williamson
Keyword(s):  
Protein Binding ◽  
Ribosome Biogenesis ◽  
Optimal Conditions ◽  
Test Bed ◽  
Macromolecular Assembly ◽  
Bacterial Ribosome ◽  
Structural Work ◽  
Assembly Intermediate

Bacterial ribosome biogenesis has been an active area of research for more than 30 years and has served as a test-bed for the development of new biochemical, biophysical and structural techniques to understand macromolecular assembly generally. Recent work inspecting the process in vivo has advanced our understanding of the role of ribosome biogenesis factors, the co-transcriptional nature of assembly, the kinetics of the process under sub-optimal conditions, and the rRNA folding and ribosome protein binding pathways. Additionally, new structural work enabled by single-particle electron microscopy has helped to connect in vitro ribosomal protein binding maps to the underlying RNA. This review summarizes the state of these in vivo studies, provides a kinetic model for ribosome assembly under sub-optimal conditions, and describes a framework to compare newly emerging assembly intermediate structures. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals MTG1 Codes for a Conserved Protein Required for Mitochondrial Translation

2003 ◽  
Vol 14 (6) ◽  
pp. 2292-2302 ◽  
Author(s):  
Antoni Barrientos ◽  
Daniel Korr ◽  
Karen J. Barwell ◽  
Christian Sjulsen ◽  
Carl D. Gajewski ◽  
...  
Keyword(s):  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Temperature Sensitive ◽  
Large Ribosomal Subunit ◽  
Human Homologue ◽  
Mitochondrial Translation ◽  
Respiratory Deficiency ◽  
The Matrix ◽  
Mature Size

The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

scholarly journals SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaowen Wang ◽  
Hong Zhang ◽  
Russell Sapio ◽  
Jun Yang ◽  
Justin Wong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Tumor Burden ◽  
Cancer Cell Proliferation ◽  
Lung Cancer Cell ◽  
Ribosomal Subunits

AbstractSOD1 is known as the major cytoplasmic superoxide dismutase and an anticancer target. However, the role of SOD1 in cancer is not fully understood. Herein we describe the generation of an inducible Sod1 knockout in KRAS-driven NSCLC mouse model. Sod1 knockout markedly reduces tumor burden in vivo and blocks growth of KRAS mutant NSCLC cells in vitro. Intriguingly, SOD1 is enriched in the nucleus and notably in the nucleolus of NSCLC cells. The nuclear and nucleolar, not cytoplasmic, form of SOD1 is essential for lung cancer cell proliferation. Moreover, SOD1 interacts with PeBoW complex and controls its assembly necessary for pre-60S ribosomal subunit maturation. Mechanistically, SOD1 regulates co-localization of PeBoW with and processing of pre-rRNA, and maturation of cytoplasmic 60S ribosomal subunits in KRAS mutant lung cancer cells. Collectively, our study unravels a nuclear SOD1 function essential for ribosome biogenesis and proliferation in KRAS-driven lung cancer.

scholarly journals Multiple GTPases Participate in the Assembly of the Large Ribosomal Subunit in Bacillus subtilis

2006 ◽  
Vol 188 (23) ◽  
pp. 8252-8258 ◽  
Author(s):  
Laura Schaefer ◽  
William C. Uicker ◽  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Jean-Michel Jault ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Protein Composition ◽  
Large Ribosomal Subunit ◽  
Sucrose Density Gradient Centrifugation ◽  
Direct Role

ABSTRACT GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.

scholarly journals YBEY is an essential biogenesis factor for mitochondrial ribosomes

2019 ◽  
Author(s):  
Sabrina Summer ◽  
Anna Smirnova ◽  
Alessandro Gabriele ◽  
Ursula Toth ◽  
Fasemore Mandela ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ribosome Biogenesis ◽  
Specific Interaction ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Abnormal Mitochondria

ABSTRACTRibosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3’-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter recruits uS11m to the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

scholarly journals YBEY is an essential biogenesis factor for mitochondrial ribosomes

2020 ◽  
Vol 48 (17) ◽  
pp. 9762-9786 ◽  
Author(s):  
Sabrina Summer ◽  
Anna Smirnova ◽  
Alessandro Gabriele ◽  
Ursula Toth ◽  
Akinyemi Mandela Fasemore ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ribosome Biogenesis ◽  
Specific Interaction ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Abnormal Mitochondria

Abstract Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3′-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

scholarly journals Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition

2018 ◽  
Author(s):  
Jose L. Llácer ◽  
Tanweer Hussain ◽  
Adesh K. Saini ◽  
Jagpreet Nanda ◽  
Sukhvir Kaur ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Start Codon ◽  
Dual Function ◽  
Translational Initiation ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
40S Ribosomal Subunit

SUMMARYIn eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNAi in a “PIN” state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.

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