bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021–12–05
nine papers selected by
Andreas Kohler, Stockholm University
  1. The potential of mitochondrial genome engineering.
  2. Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery.
  3. Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.
  4. Malignancy and NF-κB signalling strengthen coordination between expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes.
  5. Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.
  6. METTLing in the right place: METTL8 is a mitochondrial tRNA-specific methyltransferase.
  7. Accuracy mechanism of eukaryotic ribosome translocation.
  8. Translation complex stabilization on messenger RNA and footprint profiling to study the RNA responses and dynamics of protein biosynthesis in the cells.
  9. ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function.
  1. Nat Rev Genet. 2021 Dec 02.
    The potential of mitochondrial genome engineering.
    Pedro Silva-Pinheiro, Michal Minczuk.
      Mitochondria are subject to unique genetic control by both nuclear DNA and their own genome, mitochondrial DNA (mtDNA), of which each mitochondrion contains multiple copies. In humans, mutations in mtDNA can lead to devastating, heritable, multi-system diseases that display different tissue-specific presentation at any stage of life. Despite rapid advances in nuclear genome engineering, for years, mammalian mtDNA has remained resistant to genetic manipulation, hampering our ability to understand the mechanisms that underpin mitochondrial disease. Recent developments in the genetic modification of mammalian mtDNA raise the possibility of using genome editing technologies, such as programmable nucleases and base editors, for the treatment of hereditary mitochondrial disease.
    DOI:  https://doi.org/10.1038/s41576-021-00432-x
  2. Mol Cell. 2021 Nov 19. pii: S1097-2765(21)00954-0. [Epub ahead of print]
    Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery.
    Jasmin Adriana Schäfer, Süleyman Bozkurt, Jonas Benjamin Michaelis, Kevin Klann, Christian Münch.
      Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePRODmt, a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePRODmt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.
    Keywords:  SILAC; TMT; disease; integrated stress response; mitochondria; protein translocation; proteomics; proteostasis; respiratory chain complexes; translation
    DOI:  https://doi.org/10.1016/j.molcel.2021.11.004
  3. Nucleic Acids Res. 2021 Nov 29. pii: gkab1179. [Epub ahead of print]
    Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.
    Iacopo Bicci, Claudia Calabrese, Zoe J Golder, Aurora Gomez-Duran, Patrick F Chinnery.
      Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.
    DOI:  https://doi.org/10.1093/nar/gkab1179
  4. Genome Biol. 2021 Dec 02. 22(1): 328
    Malignancy and NF-κB signalling strengthen coordination between expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes.
    Marcos Francisco Perez, Peter Sarkies.
       BACKGROUND: Mitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. The mammalian mitochondrial genome encodes for 13 mitochondrial proteins, and the remaining mitochondrial proteins are encoded by the nuclear genome. Little is known about how coordination between the expression of the two sets of genes is achieved.
    RESULTS: Correlation analysis of RNA-seq expression data from large publicly available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalisation techniques based on total read counts, such as FPKM or TPM, produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, with important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalisation (MRN) or trimmed mean of M values (TMM) methods. Using these normalisations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database.
    CONCLUSIONS: We show that a subset of healthy tissues with high expression of NF-κB show significant coordination, suggesting a role for NF-κB in ensuring balanced expression between mitochondrial and nuclear genes. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer.
    DOI:  https://doi.org/10.1186/s13059-021-02541-6
  5. Angew Chem Int Ed Engl. 2021 Dec 01.
    Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.
    Yong Woong Jun, Eddy Albarran, David L Wilson, Jun Ding, Eric T Kool.
      Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. Here we employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe ( UBER ) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.
    Keywords:  DNA damage and repair; dynamics; fluorescence probes; mitochondrial DNA
    DOI:  https://doi.org/10.1002/anie.202111829
  6. Mol Cell. 2021 Dec 02. pii: S1097-2765(21)00978-3. [Epub ahead of print]81(23): 4765-4767
    METTLing in the right place: METTL8 is a mitochondrial tRNA-specific methyltransferase.
    Eva Kowalinski, Juan D Alfonzo.
      Schöller et al. (2021) discovered that METTL8, thought of as an mRNA modifier, is a tRNA-specific mitochondrial enzyme important for mitochondrial translation and function. Paradoxically, increased expression of METTL8 is associated with high respiratory rates in pancreatic cancers.
    DOI:  https://doi.org/10.1016/j.molcel.2021.11.009
  7. Nature. 2021 Dec 01.
    Accuracy mechanism of eukaryotic ribosome translocation.
    Muminjon Djumagulov, Natalia Demeshkina, Lasse Jenner, Alexey Rozov, Marat Yusupov, Gulnara Yusupova.
      Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon-anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon-anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a 'pawl' defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation.
    DOI:  https://doi.org/10.1038/s41586-021-04131-9
  8. Crit Rev Biochem Mol Biol. 2021 Dec 01. 1-44
    Translation complex stabilization on messenger RNA and footprint profiling to study the RNA responses and dynamics of protein biosynthesis in the cells.
    Nikolay E Shirokikh.
      During protein biosynthesis, ribosomes bind to messenger (m)RNA, locate its protein-coding information, and translate the nucleotide triplets sequentially as codons into the corresponding sequence of amino acids, forming proteins. Non-coding mRNA features, such as 5' and 3' untranslated regions (UTRs), start sites or stop codons of different efficiency, stretches of slower or faster code and nascent polypeptide interactions can alter the translation rates transcript-wise. Most of the homeostatic and signal response pathways of the cells converge on individual mRNA control, as well as alter the global translation output. Among the multitude of approaches to study translational control, one of the most powerful is to infer the locations of translational complexes on mRNA based on the mRNA fragments protected by these complexes from endonucleolytic hydrolysis, or footprints. Translation complex profiling by high-throughput sequencing of the footprints allows to quantify the transcript-wise, as well as global, alterations of translation, and uncover the underlying control mechanisms by attributing footprint locations and sizes to different configurations of the translational complexes. The accuracy of all footprint profiling approaches critically depends on the fidelity of footprint generation and many methods have emerged to preserve certain or multiple configurations of the translational complexes, often in challenging biological material. In this review, a systematic summary of approaches to stabilize translational complexes on mRNA for footprinting is presented and major findings are discussed. Future directions of translation footprint profiling are outlined, focusing on the fidelity and accuracy of inference of the native in vivo translation complex distribution on mRNA.
    Keywords:  RNA sequencing; Translation complex profile sequencing; high-throughput methods; ribosome; ribosome profiling; translational control
    DOI:  https://doi.org/10.1080/10409238.2021.2006599
  9. Commun Biol. 2021 Dec 02. 4(1): 1350
    ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function.
    Adrián Cortés Sanchón, Harshitha Santhosh Kumar, Matilde Mantovani, Ivan Osinnii, José María Mateos, Andres Kaech, Dimitri Shcherbakov, Rashid Akbergenov, Erik C Böttger.
      Proteostasis is a challenge for cellular organisms, as all known protein synthesis machineries are error-prone. Here we show by cell fractionation and microscopy studies that misfolded proteins formed in the endoplasmic reticulum can become associated with and partly transported into mitochondria, resulting in impaired mitochondrial function. Blocking the endoplasmic reticulum-mitochondria encounter structure (ERMES), but not the mitochondrial sorting and assembly machinery (SAM) or the mitochondrial surveillance pathway components Msp1 and Vms1, abrogated mitochondrial sequestration of ER-misfolded proteins. We term this mitochondria-associated proteostatic mechanism for ER-misfolded proteins ERAMS (ER-associated mitochondrial sequestration). We testify to the relevance of this pathway by using mutant α-1-antitrypsin as an example of a human disease-related misfolded ER protein, and we hypothesize that ERAMS plays a role in pathological features such as mitochondrial dysfunction.
    DOI:  https://doi.org/10.1038/s42003-021-02873-w
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