bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021‒06‒20
twelve papers selected by
Andreas Aufschnaiter
Stockholm University

  1. Nat Commun. 2021 06 16. 12(1): 3671
      Mitochondrial 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.
  2. Nat Commun. 2021 06 16. 12(1): 3673
      Mitochondrial 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.
  3. Mol Genet Metab. 2021 Jun 10. pii: S1096-7192(21)00721-6. [Epub ahead of print]
      Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder.
    Keywords:  Cardiomyopathy; Combined deficiency; Genetic cause; Mitochondrial ribosome; Mitochondrial translation
  4. J Biol Chem. 2021 Jun 10. pii: S0021-9258(21)00669-4. [Epub ahead of print] 100869
      Pentatricopeptide repeat (PPR) proteins are a large family of proteins that act primarily at different post-transcriptional steps of organellar gene expression. We have previously found that the Schizosaccharomyces pombe PPR protein Ppr10 interacts with mitochondrial translational activator Mpa1, and both are essential for mitochondrial protein synthesis. However, it is unclear how these two proteins function in mitochondrial protein synthesis in S. pombe. In this study, we further investigated the role of Ppr10 and Mpa1 in mitochondrial protein synthesis. Mitochondrial translational initiation requires two initiation factors, Mti2 and Mti3, which bind to the small subunit of the mitochondrial ribosome (mt-SSU) during the formation of the mitochondrial translational initiation complex. Using sucrose gradient sedimentation analysis, we found that disruption of ppr10, mpa1 or the PPR motifs in Ppr10 impairs the association of Mti2 and Mti3 with the mt-SSU, suggesting that both Ppr10 and Mpa1 may be required for the interaction of Mti2 and Mti3 with the mt-SSU during the assembly of mitochondrial translational initiation complex. Loss of Ppr10 perturbs the association of mitochondrially encoded cytochrome b (cob1) and cytochrome c oxidase subunit 1 (cox1) mRNAs with assembled mitochondrial ribosomes. Proteomic analysis revealed that a fraction of Ppr10 and Mpa1 copurified with a subset of mitoribosomal proteins. The PPR motifs of Ppr10 are necessary for its interaction with Mpa1 and that disruption of these PPR motifs impairs mitochondrial protein synthesis. Our results suggest that Ppr10 and Mpa1 function together to mediate mitochondrial translational initiation.
    Keywords:  Mpa1; Mti2; Mti3; PPR motif; Ppr10; Schizosaccharomyces pombe; mitochondrial translation
  5. Nat Commun. 2021 06 16. 12(1): 3672
      Ribosome 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.
  6. Nat Commun. 2021 06 14. 12(1): 3607
      Ribosomes 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.
  7. Environ Mol Mutagen. 2021 Jun 11.
      Digital PCR (dPCR) has been adapted to quantify the proportion of mitochondrial DNA (mtDNA) molecules without and with double-strand DNA breaks (DSBs). This is based on a break-apart approach of two differentially labeled target sequences distantly located in the circular DNA molecule. When the two targets amplify in separated reaction partitions, the original mtDNA molecule should be fragmented by two DSBs at least, each in a different segment between targets. When both targets amplify in the same partition, it must correspond to a circular or linear mtDNA molecule. These two possibilities may be distinguished through a restriction endonuclease (RE) induced unique DSB within a DNA segment between the targets. After RE-digestion, separation of both target signals in different partitions must indicate the presence of a previous linear mtDNA molecule. Otherwise, joint amplification in the same partition would correspond to an initial circular mtDNA that has been linearized by the endonuclease. The procedure was validated by assaying different proportions of mtDNA fragmented by in vitro digestion with REs, evidencing a perfect accordance between the expected theoretical values and dPCR quantification. Samples from peripheral blood cells, cellular and extracellular DNA from the U2OS cell line, as well as cells incubated with ethidium bromide to induce mtDNA depletion, were evaluated. The technique may be of interest to complement the studies of mtDNA in relation to aging and human disease, as well as to assess possible adverse effects of certain drugs that could be related to affectation of mtDNA.
    Keywords:  DNA fragmentation; digital PCR; double-strand DNA breaks (DSBs); mitochondrial DNA
  8. Crit Rev Biochem Mol Biol. 2021 Jun 13. 1-16
      Heteroplasmy refers to the coexistence of more than one variant of the mitochondrial genome (mtDNA). Mutated or partially deleted mtDNAs can induce chronic metabolic impairment and cause mitochondrial diseases when their heteroplasmy levels exceed a critical threshold. These mutant mtDNAs can be maternally inherited or can arise de novo. Compelling evidence has emerged showing that mutant mtDNA levels can vary and change in a nonrandom fashion across generations and amongst tissues of an individual. However, our lack of understanding of the basic cellular and molecular mechanisms of mtDNA heteroplasmy dynamics has made it difficult to predict who will inherit or develop mtDNA-associated diseases. More recently, with the advances in technology and the establishment of tractable model systems, insights into the mechanisms underlying the selection forces that modulate heteroplasmy dynamics are beginning to emerge. In this review, we summarize evidence from different organisms, showing that mutant mtDNA can experience both positive and negative selection. We also review the recently identified mechanisms that modulate heteroplasmy dynamics. Taken together, this is an opportune time to survey the literature and to identify key cellular pathways that can be targeted to develop therapies for diseases caused by heteroplasmic mtDNA mutations.
    Keywords:  Heteroplasmy dynamics; mitochondria; mitochondrial genetics; mtDNA; selection
  9. Sci Rep. 2021 Jun 16. 11(1): 12726
      mtDNA recombination events in yeasts are known, but altered mitochondrial genomes were not completed. Therefore, we analyzed recombined mtDNAs in six Saccharomyces cerevisiae × Saccharomyces paradoxus hybrids in detail. Assembled molecules contain mostly segments with variable length introgressed to other mtDNA. All recombination sites are in the vicinity of the mobile elements, introns in cox1, cob genes and free standing ORF1, ORF4. The transplaced regions involve co-converted proximal exon regions. Thus, these selfish elements are beneficial to the host if the mother molecule is challenged with another molecule for transmission to the progeny. They trigger mtDNA recombination ensuring the transfer of adjacent regions, into the progeny of recombinant molecules. The recombination of the large segments may result in mitotically stable duplication of several genes.
  10. Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2101164118. [Epub ahead of print]118(25):
      While RNAs are known to misfold, the underlying molecular causes have been mainly studied in fragments of biologically relevant larger RNAs. As these small RNAs are dominated by secondary structures, misfolding of these secondary structures remains the most-explored cause for global RNA misfolding. Conversely, how RNA chaperones function in a biological context to promote native folding beyond duplex annealing remains unknown. Here, in a combination of dimethylsulfate mutational profiling with sequencing (DMS-MaPseq), structural analyses, biochemical experiments, and yeast genetics, we show that three-helix junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of premature tertiary interactions, which otherwise kinetically trap misfolded junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners of junctions, our analyses also suggest direct roles for small nucleolar RNAs (snoRNAs) in binding and chaperoning helical junctions during transcription. While these assembly factors do not utilize energy to ameliorate misfolding, our data demonstrate how their dissociation renders reversible folding steps irreversible, thereby driving native folding and assembly and setting up a timer that dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate that RNA chaperones act locally on individual tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.
    Keywords:  RNA chaperone; RNA folding; helix junction; ribosome assembly
  11. Cell Syst. 2021 Jun 16. pii: S2405-4712(21)00110-1. [Epub ahead of print]12(6): 593-607
      Molecular translation systems provide a genetically encoded framework for protein synthesis, which is essential for all life. Engineering these systems to incorporate non-canonical amino acids (ncAAs) into peptides and proteins has opened many exciting opportunities in chemical and synthetic biology. Here, we review recent advances that are transforming our ability to engineer molecular translation systems. In cell-based systems, new processes to synthesize recoded genomes, tether ribosomal subunits, and engineer orthogonality with high-throughput workflows have emerged. In cell-free systems, adoption of flexizyme technology and cell-free ribosome synthesis and evolution platforms are expanding the limits of chemistry at the ribosome's RNA-based active site. Looking forward, innovations will deepen understanding of molecular translation and provide a path to polymers with previously unimaginable structures and functions.
    Keywords:  chemical biology; non-canonical amino acids; orthogonal translation systems; protein synthesis; synthetic biology; systems biology
  12. Nucleic Acids Res. 2021 Jun 14. pii: gkab495. [Epub ahead of print]
      How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.