Maintaining mitochondrial ribosome function: The role of ribosome rescue and recycling factors

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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Mitochondrial microRNAs: A Putative Role in Tissue Regeneration

Biology ◽

10.3390/biology9120486 ◽

2020 ◽

Vol 9 (12) ◽

pp. 486

Author(s):

Sílvia C. Rodrigues ◽

Renato M. S. Cardoso ◽

Filipe V. Duarte

Keyword(s):

Tissue Regeneration ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Noncoding Rnas ◽

Atp Synthesis ◽

Mitochondrial Proteins ◽

Cellular Mechanisms ◽

Small Noncoding Rnas ◽

Mitochondrial Ribosome

The most famous role of mitochondria is to generate ATP through oxidative phosphorylation, a metabolic pathway that involves a chain of four protein complexes (the electron transport chain, ETC) that generates a proton-motive force that in turn drives the ATP synthesis by the Complex V (ATP synthase). An impressive number of more than 1000 mitochondrial proteins have been discovered. Since mitochondrial proteins have a dual genetic origin, it is predicted that ~99% of these proteins are nuclear-encoded and are synthesized in the cytoplasmatic compartment, being further imported through mitochondrial membrane transporters. The lasting 1% of mitochondrial proteins are encoded by the mitochondrial genome and synthesized by the mitochondrial ribosome (mitoribosome). As a result, an appropriate regulation of mitochondrial protein synthesis is absolutely required to achieve and maintain normal mitochondrial function. Regarding miRNAs in mitochondria, it is well-recognized nowadays that several cellular mechanisms involving mitochondria are regulated by many genetic players that originate from either nuclear- or mitochondrial-encoded small noncoding RNAs (sncRNAs). Growing evidence collected from whole genome and transcriptome sequencing highlight the role of distinct members of this class, from short interfering RNAs (siRNAs) to miRNAs and long noncoding RNAs (lncRNAs). Some of the mechanisms that have been shown to be modulated are the expression of mitochondrial proteins itself, as well as the more complex coordination of mitochondrial structure and dynamics with its function. We devote particular attention to the role of mitochondrial miRNAs and to their role in the modulation of several molecular processes that could ultimately contribute to tissue regeneration accomplishment.

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The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential

Human Molecular Genetics ◽

10.1093/hmg/ddt490 ◽

2013 ◽

Vol 23 (4) ◽

pp. 949-967 ◽

Cited By ~ 20

Author(s):

Paul M. Smith ◽

Joanna L. Elson ◽

Laura C. Greaves ◽

Saskia B. Wortmann ◽

Richard J.T. Rodenburg ◽

...

Keyword(s):

Human Disease ◽

Mitochondrial Rrna ◽

Mitochondrial Ribosome

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The post-transcriptional life of mammalian mitochondrial RNA

Biochemical Journal ◽

10.1042/bj20112208 ◽

2012 ◽

Vol 444 (3) ◽

pp. 357-373 ◽

Cited By ~ 87

Author(s):

Joanna Rorbach ◽

Michal Minczuk

Keyword(s):

Messenger Rna ◽

Mitochondrial Gene ◽

Rna Stability ◽

Stability Control ◽

Mitochondrial Rna ◽

Mammalian Mitochondria ◽

Mitochondrial Ribosome ◽

Rna Maturation ◽

Rna Polyadenylation

Mammalian mitochondria contain their own genome that encodes mRNAs for thirteen essential subunits of the complexes performing oxidative phosporylation as well as the RNA components (two rRNAs and 22 tRNAs) needed for their translation in mitochondria. All RNA species are produced from single polycistronic precursor RNAs, yet the relative concentrations of various RNAs differ significantly. This underscores the essential role of post-transcriptional mechanisms that control the maturation, stability and translation of mitochondrial RNAs. The present review provides a detailed summary on the role of RNA maturation in the regulation of mitochondrial gene expression, focusing mainly on messenger RNA polyadenylation and stability control. Furthermore, the role of mitochondrial ribosomal RNA stability, processing and modifications in the biogenesis of the mitochondrial ribosome is discussed.

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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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Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance

Nature Communications ◽

10.1038/s41467-021-23726-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ravi Kiran Koripella ◽

Ayush Deep ◽

Ekansh K. Agrawal ◽

Pooja Keshavan ◽

Nilesh K. Banavali ◽

...

Keyword(s):

Messenger Rna ◽

Elongation Factor ◽

Acid Resistance ◽

Functional Specificity ◽

Ribosomal Subunits ◽

Sequence Of Events ◽

Mitochondrial Ribosome ◽

Distinct Sequence ◽

Mitochondrial Elongation

AbstractRibosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt). These structures clarify an unusual role of a mitochondria-specific segment of RRFmt, identify the structural distinctions that confer functional specificity to EF-G2mt, and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2mt is markedly different from that of mitochondrial elongation factor EF-G1mt, suggesting that the two human EF-Gmts have evolved diversely to negate the effect of a bacterial antibiotic.

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Visualising formation of the ribosomal active site in mitochondria

10.1101/2021.03.19.436169 ◽

2021 ◽

Author(s):

Viswanathan Chandrasekaran ◽

Nirupa Desai ◽

Nicholas O. Burton ◽

Hanting Yang ◽

Jon Price ◽

...

Keyword(s):

Stress Responses ◽

Large Subunit ◽

Mitochondrial Protein ◽

Mitochondrial Stress ◽

Cryo Electron Microscopy ◽

Complex Process ◽

Mitochondrial Ribosome ◽

Peptidyl Transferase Centre

SummaryRibosome assembly is an essential and complex process that is regulated at each step by specific biogenesis factors. Using cryo-electron microscopy, we identify and order major steps in the formation of the highly conserved peptidyl transferase centre (PTC) and tRNA binding sites in the large subunit of the human mitochondrial ribosome (mitoribosome). The conserved GTPase GTPBP7 regulates the folding and incorporation of core 16S ribosomal RNA (rRNA) helices and the ribosomal protein bL36m, and ensures that the PTC base U3039 has been 2′-O-methylated. Additionally, GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which facilitate earlier steps by sequestering H68-71 of the 16S rRNA and allowing biogenesis factors to access the maturing PTC. Consistent with the central role of NSUN4•MTERF4 and GTPBP7 during mitoribosome biogenesis, in vivo mutagenesis designed to disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activates mitochondrial stress responses and results in severely reduced viability, developmental delays and sterility. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4 but not its enzymatic activity, is indispensable for mitochondrial protein synthesis in metazoans.

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Structures of the human mitochondrial ribosome recycling complexes reveal distinct mechanisms of recycling and antibiotic resistance

10.1101/2020.12.20.423689 ◽

2020 ◽

Author(s):

Ravi Kiran Koripella ◽

Ayush Deep ◽

Ekansh K. Agrawal ◽

Pooja Keshavan ◽

Nilesh K. Banavali ◽

...

Keyword(s):

Antibiotic Resistance ◽

Conformational Changes ◽

Messenger Rna ◽

Elongation Factor ◽

Ribosome Recycling Factor ◽

Ribosomal Subunits ◽

Sequence Of Events ◽

Mitochondrial Ribosome ◽

Mitochondrial Elongation

AbstractRibosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Mitochondrial ribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt) are two proteins with mitochondria-specific additional sequences that catalyze the recycling step in human mitochondria. We have determined high-resolution cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) in complex with RRFmt, and the mitoribosomal large 39S subunit in complex with both RRFmt and EF-G2mt. In addition, we have captured the structure of a short-lived intermediate state of the 55S•RRFmt•EF-G2mt complex. These structures clarify the role of a mitochondria-specific segment of RRFmt in mitoribosome recycling, identify the structural distinctions between the two isoforms of EF-Gmt that confer their functional specificity, capture recycling-specific conformational changes in the L7/L12 stalk-base region, and suggest a distinct mechanistic sequence of events in mitoribosome recycling. Furthermore, biochemical and structural assessments of the sensitivity of EF-G2mt to the antibiotic fusidic acid reveals that the molecular mechanism of antibiotic resistance for EF-G2mt is markedly different from that exhibited by mitochondrial elongation factor EF-G1mt, suggesting that these two homologous mitochondrial proteins have evolved diversely to negate the effect of a bacterial antibiotics.

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