A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

The epitranscriptome plays a key regulatory role in cellular processes in health and disease, including ribosome biogenesis. Here, analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, modified by MRM1, MRM2, and MRM3. Ablation of MRM2 leads to a severe impairment of the oxidative phosphorylation system, caused by defective mitochondrial translation and accumulation of mtLSU assembly intermediates. Structures of these particles (2.58 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module. Additionally, we present five mtLSU assembly states with different intersubunit interface configurations. Complementation studies demonstrate that the methyltransferase activity of MRM2 is dispensable for mitoribosome biogenesis. The Drosophila melanogaster orthologue, DmMRM2, is an essential gene, with its knock- down leading to developmental arrest. This work identifies a key late-stage quality control step during mtLSU assembly, ultimately contributing to the maintenance of mitochondrial homeostasis.

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Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Nucleic Acids Research ◽

10.1093/nar/gkaa1131 ◽

2020 ◽

Author(s):

Miriam Cipullo ◽

Sarah F Pearce ◽

Isabel G Lopez Sanchez ◽

Shreekara Gopalakrishna ◽

Annika Krüger ◽

...

Keyword(s):

Late Stage ◽

Large Subunit ◽

Mitochondrial Translation ◽

Gtp Hydrolysis ◽

Macromolecular Complexes ◽

Subunit Assembly ◽

Oxidative Phosphorylation System ◽

Gtp Binding ◽

Severe Impairment

Abstract Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

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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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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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Mrpl36 Is Important for Generation of Assembly Competent Proteins during Mitochondrial Translation

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1162 ◽

2009 ◽

Vol 20 (10) ◽

pp. 2615-2625 ◽

Cited By ~ 32

Author(s):

Martin Prestele ◽

Frank Vogel ◽

Andreas S. Reichert ◽

Johannes M. Herrmann ◽

Martin Ott

Keyword(s):

Protein Synthesis ◽

Respiratory Chain ◽

Large Subunit ◽

Critical Role ◽

Mitochondrial Translation ◽

Respiratory Chain Complexes ◽

Ribosomal Subunits ◽

Terminal Domain ◽

Chain Complexes ◽

Mitochondrial Ribosome

The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.

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MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

Molecular Biology of the Cell ◽

10.1091/mbc.e14-01-0014 ◽

2014 ◽

Vol 25 (17) ◽

pp. 2542-2555 ◽

Cited By ~ 55

Author(s):

Joanna Rorbach ◽

Pierre Boesch ◽

Payam A. Gammage ◽

Thomas J. J. Nicholls ◽

Sarah F. Pearce ◽

...

Keyword(s):

16S Rrna ◽

Rna Processing ◽

Large Subunit ◽

Peptidyl Transferase ◽

Mitochondrial Translation ◽

Peptidyl Transferase Center ◽

Mitochondrial Ribosome ◽

Translation Apparatus ◽

Rrna Maturation ◽

And Function

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.

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Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation

eLife ◽

10.7554/elife.59264 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Victor Tobiasson ◽

Alexey Amunts

Keyword(s):

Mitochondrial Genome ◽

Ribosomal Proteins ◽

Protein Targeting ◽

Large Subunit ◽

Model Organism ◽

Small Subunit ◽

Specific Protein ◽

Mitochondrial Translation ◽

Functional Protein ◽

Mitochondrial Ribosome

To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal 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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The Yeast GTPase Mtg2p Is Required for Mitochondrial Translation and Partially Suppresses an rRNA Methyltransferase Mutant,mrm2

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0622 ◽

2005 ◽

Vol 16 (2) ◽

pp. 954-963 ◽

Cited By ~ 43

Author(s):

Kaustuv Datta ◽

Jennifer L. Fuentes ◽

Janine R. Maddock

Keyword(s):

Mitochondrial Dna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Mitochondrial Protein ◽

Ribosomal Subunit ◽

Temporal Association ◽

Temperature Sensitive ◽

Dependent Manner ◽

Mitochondrial Translation ◽

Mitochondrial Ribosome

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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