bims-tricox Biomed News
on Translation, ribosomes and COX
Issue of 2023–01–15
six papers selected by
Yash Verma, University of Delhi South Campus
  1. Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.
  2. DPC29 promotes post-initiation mitochondrial translation in Saccharomyces cerevisiae.
  3. Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP.
  4. A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle.
  5. Ready, steady, go: Rapid ribosomal scanning to reach start codons.
  6. pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.
  1. Nucleic Acids Res. 2023 Jan 11. pii: gkac1233. [Epub ahead of print]
    Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.
    Cristina Remes, Anas Khawaja, Sarah F Pearce, Adam M Dinan, Shreekara Gopalakrishna, Miriam Cipullo, Vasileios Kyriakidis, Jingdian Zhang, Xaquin Castro Dopico, Olessya Yukhnovets, Ilian Atanassov, Andrew E Firth, Barry Cooperman, Joanna Rorbach.
      The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.
    DOI:  https://doi.org/10.1093/nar/gkac1233
  2. Nucleic Acids Res. 2023 Jan 09. pii: gkac1229. [Epub ahead of print]
    DPC29 promotes post-initiation mitochondrial translation in Saccharomyces cerevisiae.
    Kyle A Hubble, Michael F Henry.
      Mitochondrial ribosomes synthesize essential components of the oxidative phosphorylation (OXPHOS) system in a tightly regulated process. In the yeast Saccharomyces cerevisiae, mitochondrial mRNAs require specific translational activators, which orchestrate protein synthesis by recognition of their target gene's 5'-untranslated region (UTR). Most of these yeast genes lack orthologues in mammals, and only one such gene-specific translational activator has been proposed in humans-TACO1. The mechanism by which TACO1 acts is unclear because mammalian mitochondrial mRNAs do not have significant 5'-UTRs, and therefore must promote translation by alternative mechanisms. In this study, we examined the role of the TACO1 orthologue in yeast. We found this 29 kDa protein to be a general mitochondrial translation factor, Dpc29, rather than a COX1-specific translational activator. Its activity was necessary for the optimal expression of OXPHOS mtDNA reporters, and mutations within the mitoribosomal large subunit protein gene MRP7 produced a global reduction of mitochondrial translation in dpc29Δ cells, indicative of a general mitochondrial translation factor. Northern-based mitoribosome profiling of dpc29Δ cells showed higher footprint frequencies at the 3' ends of mRNAs, suggesting a role in translation post-initiation. Additionally, human TACO1 expressed at native levels rescued defects in dpc29Δ yeast strains, suggesting that the two proteins perform highly conserved functions.
    DOI:  https://doi.org/10.1093/nar/gkac1229
  3. Bio Protoc. 2022 Dec 20. pii: e4578. [Epub ahead of print]12(24):
    Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP.
    Jonas B Michaelis, Süleyman Bozkurt, Jasmin A Schäfer, Christian Münch.
      Mitochondria are cellular organelles essential for the function and survival of eukaryotic cells. Nearly all mitochondrial proteins are nuclear-encoded and require mitochondrial import upon their synthesis in the cytosol. Various approaches have been described to study mitochondrial protein import, such as monitoring the entry of radiolabeled proteins into purified mitochondria or quantifying newly synthesized proteins within mitochondria by proteomics. Here, we provide a detailed protocol for a commonly used and straightforward assay that quantitatively examines mitochondrial protein import by monitoring the co-localization of mitochondrially targeted enhanced green fluorescent protein (eGFP) with the mitochondrial fluorescence dye MitoTracker TM Deep Red FM by live cell imaging. We describe the preparation and use of a stable mammalian cell line inducibly expressing a mitochondrial targeting sequence (MTS)-eGFP, followed by quantitative image analysis using an open-source ImageJ-based plugin. This inducible expression system avoids the need for transient transfection while enabling titration of MTS-eGFP expression and thereby avoiding protein folding stress. Overall, the assay provides a simple and robust approach to assess mitochondrial import capacity of cells in various disease-related settings. This protocol was validated in: Mol Cell (2021), DOI: 10.1016/j.molcel.2021.11.004 Graphical abstract.
    Keywords:   Live cell imaging ; Microscopy ; Mitochondria ; Mitochondrial protein import ; Protein translocation
    DOI:  https://doi.org/10.21769/BioProtoc.4578
  4. Open Biol. 2023 Jan;13(1): 220238
    A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle.
    Yi-Kai Fang, Zuzana Vaitová, Vladimir Hampl.
      The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.
    Keywords:  evolution of protein targeting; hydrogenosome; mitochondrion-free eukaryote; protein import
    DOI:  https://doi.org/10.1098/rsob.220238
  5. Mol Cell. 2023 Jan 05. pii: S1097-2765(22)01165-0. [Epub ahead of print]83(1): 9-11
    Ready, steady, go: Rapid ribosomal scanning to reach start codons.
    Helge Paternoga, Daniel N Wilson.
      Wang et al. (2022)1 employ real-time single-molecule fluorescence spectroscopy to monitor eukaryotic translation initiation events, revealing that, while mRNA engagement by ribosomal 43S subunits is slow, the subsequent mRNA scanning process is rapid- ∼10 times faster than translation.
    DOI:  https://doi.org/10.1016/j.molcel.2022.12.008
  6. FEMS Yeast Res. 2023 Jan 14. pii: foad002. [Epub ahead of print]
    pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.
    Letizia Maestroni, Pietro Butti, Vittorio Giorgio Senatore, Paola Branduardi.
      CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted Double Strand Breaks (DSB) is controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome-editing in S. cerevisiae, pCEC-red. This tool allows i) to transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and ii) to insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (close to 90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.
    Keywords:   ADE2 deletion; Saccharomyces cerevisiae ; CRISPR-Cas9; genome editing; guide RNA; new plasmid
    DOI:  https://doi.org/10.1093/femsyr/foad002
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