bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021‒06‒27
eleven papers selected by
Andreas Aufschnaiter
Stockholm University

  1. Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2023752118. [Epub ahead of print]118(25):
      Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.
    Keywords:  T cell; fever; immunology; metabolism; mitochondria
  2. Hum Mol Genet. 2021 Jun 24. pii: ddab156. [Epub ahead of print]
      Mitochondrial DNA (mtDNA) disorders are recognised as one of the most common causes of inherited metabolic disorders. The mitochondrial genome occurs in multiple copies resulting in both homoplasmic and heteroplasmic pathogenic mtDNA variants. A biochemical defect arises when the pathogenic variant level reaches a threshold, which differs between variants. Moreover, variants can segregate, clonally expand, or be lost from cellular populations resulting in a dynamic and tissue-specific mosaic pattern of oxidative deficiency. MtDNA is maternally inherited but transmission patterns of heteroplasmic pathogenic variants are complex. During oogenesis, a mitochondrial bottleneck results in offspring with widely differing variant levels to their mother, whilst highly deleterious variants, such as deletions, are not transmitted. Complemented by a complex interplay between mitochondrial and nuclear genomes, these peculiar genetics produce marked phenotypic variation, posing challenges to the diagnosis and clinical management of patients. Novel therapeutic compounds and several genetic therapies are currently under investigation, but proven disease-modifying therapies remain elusive. Women who carry pathogenic mtDNA variants require bespoke genetic counselling to determine their reproductive options. Recent advances in in vitro fertilisation techniques, have greatly improved reproductive choices, but are not without their challenges. Since the first pathogenic mtDNA variants were identified over thirty years ago, there has been remarkable progress in our understanding of these diseases. However, many questions remain unanswered and future studies are required to investigate the mechanisms of disease progression and to identify new disease-specific therapeutic targets.
  3. Mol Microbiol. 2021 Jun 21.
      Most bacteria are quiescent, typically as a result of nutrient limitation. In order to minimize energy consumption during this potentially prolonged state, quiescent bacteria substantially attenuate protein synthesis, the most energetically costly cellular process. Ribosomes in quiescent bacteria are present as dimers of two 70S ribosomes. Dimerization is dependent on a single protein, hibernation promoting factor (HPF), that binds the ribosome in the mRNA channel. This interaction indicates that dimers are inactive, suggesting that HPF inhibits translation. However, we observe that HPF does not significantly affect protein synthesis in vivo suggesting that dimerization is a consequence of inactivity, not the cause. The HPF-dimer interaction further implies that re-initiation of translation when the bacteria exit quiescence requires dimer resolution. We show that ribosome dimers quickly resolve in the presence of nutrients, and this resolution is dependent on transcription, indicating that mRNA synthesis is required for dimer resolution. Finally, we observe that ectopic HPF expression in growing cells where mRNA is abundant does not significantly affect protein synthesis despite stimulating dimer formation, suggesting that dimerization is dynamic. Thus, the extensive transcription that occurs in response to nutrient availability rapidly re-activates the translational apparatus of a quiescent cell and induces dimer resolution.
    Keywords:  protein synthesis; ribosome hibernation; stationary phase; translation
  4. BMC Bioinformatics. 2021 Jun 19. 22(1): 336
      BACKGROUND: With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays.RESULTS: Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5'UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5' UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions.
    CONCLUSION: In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment.
    Keywords:  5′ UTRs; Analysis workflow; CAGE; Open reading frames; RNA-seq; Ribo-seq; TCP-seq; Translation; Translation initiation; uORFs
  5. Nat Commun. 2021 06 22. 12(1): 3850
      Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.
  6. Nucleic Acids Res. 2021 Jun 22. pii: gkab532. [Epub ahead of print]
      Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
  7. Nucleic Acids Res. 2021 Jun 23. pii: gkab522. [Epub ahead of print]
      Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.
  8. Proc Natl Acad Sci U S A. 2021 Jun 29. pii: e2100474118. [Epub ahead of print]118(26):
      During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.
    Keywords:  YidC; membrane proteins; ribosome; single-molecule biophysics; translocon SecYEG
  9. J Phys Chem B. 2021 Jun 24. 125(24): 6543-6558
      This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.
  10. Theranostics. 2021 ;11(15): 7337-7359
      Background: Hypoxia is a hallmark of the physical microenvironment of solid tumors. As a key factor that regulates tumor development and progression, hypoxia can reprogram the expression of multiple genes, whose biological function and molecular mechanism in cancer remain largely unclear. The mitochondrial ribosome protein family consists of nuclear-encoded mitochondrial proteins that are responsible for protein synthesis in the mitochondria. Methods: A high-throughput RNA sequencing assay was carried out to identify differentially expressed mRNAs between breast cancer tissues and adjacent normal tissues as well as breast tumors with metastasis and those without metastasis. Our clinical samples and TCGA database were analyzed to observe the clinical value of mitochondrial ribosome protein L52 (MRPL52) in human breast cancer. Potent hypoxia response elements in the promoter region of MRPL52 were identified and validated by chromatin immunoprecipitation and luciferase reporter assays. Functional experiments were performed using breast cancer cell lines with MRPL52 ectopic expression and knockdown cultured in a 20% or 1% O2 environment. Results: MRPL52 expression was upregulated in human breast cancer and was significantly associated with aggressive clinicopathological characteristics and a higher metastatic risk of breast cancer patients. We found that the overexpression of MRPL52 in breast cancer is induced by hypoxia-inducible factor-1 in response to hypoxic exposure. The role of MRPL52 in suppressing apoptosis and promoting migration and invasion of hypoxic breast cancer cells was demonstrated by our experimental evidence. Mechanistically, MRPL52 promoted PTEN-induced putative kinase 1 /Parkin-dependent mitophagy to remove oxidatively damaged mitochondria and prevent uncontrolled reactive oxygen species (ROS) generation, thus repressing activation of the mitochondrial apoptotic cascade. Additionally, MRPL52 augmented epithelial-mesenchymal transition, migration and invasion of hypoxic breast cancer cells by activating the ROS-Notch1-Snail signaling pathway. Benefited from this bidirectional regulatory mechanism, MRPL52 is responsible for maintaining ROS levels in a window that can induce tumorigenic signal transduction without causing cytotoxicity in hypoxic breast cancer cells. Conclusions: This work elucidates the molecular mechanism by which MRPL52 mediates hypoxia-induced apoptotic resistance and metastatic initiation of breast cancer, and provides new insights into the interplay between cancer and the tumor microenvironment.
    Keywords:  Breast cancer; Hypoxia; Metastasis; Mitochondrial ribosome; Mitophagy
  11. Curr Protoc. 2021 Jun;1(6): e176
      RNA sequencing (RNA-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total RNA is ribosomal RNA (rRNA). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger RNA (mRNA) and long noncoding RNAs (lncRNA), it is essential to minimize the rRNA being sequenced. Although commercial methods exist to deplete rRNA from total RNA samples before sequencing, they are expensive and require specific amounts of input RNA, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rRNA depletion protocol using RNase H and DNA oligonucleotides complementary to human rRNA transcripts. This protocol includes guidelines for DNA oligo preparation, RNA:DNA hybridization, RNase H cleavage and RNA cleanup, and benchmarking of rRNA depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rRNA depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input RNA amounts. © 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rRNA transcripts from human total RNA Support Protocol: Preparation of the rRNA depletion DNA oligonucleotide pool.
    Keywords:  RNA-seq; mRNA; noncoding RNA; ribosomal RNA