The Yeast GTPase Mtg2p Is Required for Mitochondrial Translation and Partially Suppresses an rRNA Methyltransferase Mutant,mrm2

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.

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IRC3 regulates mitochondrial translation in response to metabolic cues in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.00233-21 ◽

2021 ◽

Author(s):

Jaswinder Kaur ◽

Kaustuv Datta

Keyword(s):

Mitochondrial Dna ◽

Mitochondrial Protein ◽

Ribosomal Subunit ◽

Growth Defect ◽

Temperature Sensitive ◽

Reporter System ◽

Mitochondrial Translation ◽

Source Reduction ◽

Translation Elongation ◽

Dna Maintenance

Mitochondrial oxidative phosphorylation (OXPHOS) enzymes are made up of dual genetic origin. Mechanisms regulating the expression of nuclear-encoded OXPHOS subunits in response to metabolic cues (glucose vs. glycerol), is significantly understood while regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3 a DEAD/H box helicase, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunit in cells consistent with its role in regulating translation elongation based on Arg8 m reporter system. IRC3 deleted cells retained mitochondrial DNA despite growth defect on glycerol plates. Glucose grown Δirc3ρ + and irc3 temperature-sensitive cells at 37 0 C have reduced translation rates from majority of mRNAs. In contrast, when galactose was the carbon source, reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ + but no defect was observed in irc3 temperature-sensitive cells, at 37 0 C. In support, of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, suppressors of Δirc3 isolated for restoration of growth on glycerol media restore mitochondrial protein synthesis differentially in presence of glucose vs. glycerol.

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Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Nature Communications ◽

10.1038/s41467-021-23702-y ◽

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.

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MTG1 Codes for a Conserved Protein Required for Mitochondrial Translation

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0636 ◽

2003 ◽

Vol 14 (6) ◽

pp. 2292-2302 ◽

Cited By ~ 45

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.

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Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

The Journal of Cell Biology ◽

10.1083/jcb.95.1.267 ◽

1982 ◽

Vol 95 (1) ◽

pp. 267-277 ◽

Cited By ~ 3

Author(s):

R J Lapolla ◽

A M Lambowitz

Keyword(s):

Ribosomal Proteins ◽

Mitochondrial Protein ◽

Ribosomal Subunit ◽

Small Subunit ◽

Ribosome Assembly ◽

Ribosomal Subunits ◽

Mitochondrial Ribosome ◽

Equilibrium Centrifugation ◽

Insight Into

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

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YBEY is an essential biogenesis factor for mitochondrial ribosomes

10.1101/2019.12.13.874362 ◽

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.

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YBEY is an essential biogenesis factor for mitochondrial ribosomes

Nucleic Acids Research ◽

10.1093/nar/gkaa148 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9762-9786 ◽

Cited By ~ 3

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.

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The conserved interaction of C7orf30 with MRPL14 promotes biogenesis of the mitochondrial large ribosomal subunit and mitochondrial translation

Molecular Biology of the Cell ◽

10.1091/mbc.e12-09-0651 ◽

2013 ◽

Vol 24 (3) ◽

pp. 184-193 ◽

Cited By ~ 27

Author(s):

Stephen Fung ◽

Tamiko Nishimura ◽

Florin Sasarman ◽

Eric A. Shoubridge

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Large Family ◽

Large Ribosomal Subunit ◽

Mitochondrial Translation ◽

Interacting Protein ◽

Mitochondrial Ribosome ◽

Essential Components ◽

Translation Apparatus ◽

Interfering Rna

Mammalian mitochondria harbor a dedicated translation apparatus that is required for the synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides, all of which are essential components of the oxidative phosphorylation (OXPHOS) complexes. Little is known about the mechanism of assembly of the mitoribosomes that catalyze this process. Here we show that C7orf30, a member of the large family of DUF143 proteins, associates with the mitochondrial large ribosomal subunit (mt-LSU). Knockdown of C7orf30 by short hairpin RNA (shRNA) does not alter the sedimentation profile of the mt-LSU, but results in the depletion of several mt-LSU proteins and decreased monosome formation. This leads to a mitochondrial translation defect, involving the majority of mitochondrial polypeptides, and a severe OXPHOS assembly defect. Immunoprecipitation and mass spectrometry analyses identified mitochondrial ribosomal protein (MRP)L14 as the specific interacting protein partner of C7orf30 in the mt-LSU. Reciprocal experiments in which MRPL14 was depleted by small interfering RNA (siRNA) phenocopied the C7orf30 knockdown. Members of the DUF143 family have been suggested to be universally conserved ribosomal silencing factors, acting by sterically inhibiting the association of the small and large ribosomal subunits. Our results demonstrate that, although the interaction between C7orf30 and MRPL14 has been evolutionarily conserved, human C7orf30 is, on the contrary, essential for mitochondrial ribosome biogenesis and mitochondrial translation.

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Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

10.1101/2021.03.17.435767 ◽

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.

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Requirements for the nuclear export of the small ribosomal subunit

Journal of Cell Science ◽

10.1242/jcs.115.14.2985 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2985-2995 ◽

Cited By ~ 4

Author(s):

Terence I. Moy ◽

Pamela A. Silver

Keyword(s):

Nuclear Export ◽

Ribosome Biogenesis ◽

Large Scale ◽

Ribosomal Subunit ◽

Small Subunit ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Small Ribosomal Subunit ◽

Factors Affecting ◽

The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

mBio ◽

10.1128/mbio.00029-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 17

Author(s):

S. Karniely ◽

M. P. Weekes ◽

R. Antrobus ◽

J. Rorbach ◽

L. van Haute ◽

...

Keyword(s):

Tumor Cells ◽

Human Cytomegalovirus ◽

Mitochondrial Biogenesis ◽

Ribosome Biogenesis ◽

Hcmv Infection ◽

Aerobic Glycolysis ◽

Tricarboxylic Acid Cycle ◽

Mitochondrial Translation ◽

Mitochondrial Transcription ◽

Mitochondrial Ribosome

ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. IMPORTANCE Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

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