bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021‒08‒01
ten papers selected by
Andreas Aufschnaiter
Stockholm University


  1. Nat Commun. 2021 07 27. 12(1): 4544
      Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.
    DOI:  https://doi.org/10.1038/s41467-021-24818-x
  2. Nat Chem. 2021 Aug;13(8): 751-757
      The translation of messenger RNA sequences into polypeptide sequences according to the genetic code is central to life. How this process, which relies on the ribosomal machinery, arose from much simpler precursors is unclear. Here, we demonstrate that single nucleotides charged with an amino acid couple with amino acids linked to the 5'-terminus of an RNA primer in reactions directed by the nucleotides of an RNA template in dilute aqueous solution at 0 °C. When a mixture of U-Val, A-Gly and G-Leu competed for coupling to Gly-RNA, base pairing dictated which dipeptide sequence formed preferentially. The resulting doubly anchored dipeptides can retain their link to the primer for further extension or can be fully released under mild acidic conditions. These results show that a single-nucleotide-based form of translation exists that requires no more than oligoribonucleotides and anchored amino acids.
    DOI:  https://doi.org/10.1038/s41557-021-00749-4
  3. Trends Biochem Sci. 2021 Jul 23. pii: S0968-0004(21)00144-4. [Epub ahead of print]
      The conceptual origins of ribosome specialization can be traced back to the earliest days of molecular biology. Yet, this field has only recently begun to gather momentum, with numerous studies identifying distinct heterogeneous ribosome populations across multiple species and model systems. It is proposed that some of these compositionally distinct ribosomes may be functionally specialized and able to regulate the translation of specific mRNAs. Identification and functional characterization of specialized ribosomes has the potential to elucidate a novel layer of gene expression control, at the level of translation, where the ribosome itself is a key regulatory player. In this review, we discuss different sources of ribosome heterogeneity, evidence for ribosome specialization, and also the future directions of this exciting field.
    Keywords:  rRNA modifications; ribosomal RNA (rRNA); ribosomal proteins (RPs); ribosomopathies; specialized ribosomes
    DOI:  https://doi.org/10.1016/j.tibs.2021.07.001
  4. Nat Commun. 2021 Jul 30. 12(1): 4644
      Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.
    DOI:  https://doi.org/10.1038/s41467-021-24911-1
  5. J Vis Exp. 2021 Jul 06.
      The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.
    DOI:  https://doi.org/10.3791/62664
  6. Cell. 2021 Jul 19. pii: S0092-8674(21)00831-X. [Epub ahead of print]
      Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 d RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
    Keywords:  ADP-ribosylation; MARylation; NAD(+); NAD(+) sensor; NAD(+) synthesis; NMNAT-2; PARP-16; cancer; mRNA translation; mono(ADP-ribose); mono(ADP-ribosyl)ation; ovarian cancer; protein aggregation; protein synthesis; ribosomes; translation
    DOI:  https://doi.org/10.1016/j.cell.2021.07.005
  7. Elife. 2021 Jul 28. pii: e70560. [Epub ahead of print]10
      In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.
    Keywords:  biochemistry; cell biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.70560
  8. eNeuro. 2021 Jul 26. pii: ENEURO.0232-21.2021. [Epub ahead of print]
      Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single cell mRNA sequencing could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained from microdissected brain tissue segregated the hippocampus, striatum, and cortex from each other. Nuclear-encoded mitochondrial transcripts could only segregate cell types and brain regions when the analysis was performed at the single cell level. In fact, single cell mitochondrial transcriptomes were able to distinguish glutamatergic and distinct types of GABAergic neurons from one another. Within these cell categories, unique SLC25A transporters were able to identify distinct cell subpopulations. Our results demonstrate heterogeneous mitochondrial composition across brain regions and cell types. We postulate that mitochondrial heterogeneity influences regional and cell type specific mechanisms in health and disease.Significance StatementMitochondria are important organelles for maintaining brain health. The composition of proteins making up mitochondria is essential for their function. Disturbances to mitochondria are thought to contribute to neurodegeneration and neurodevelopmental disorders. These conditions typically affect specific brain regions or cell types. Despite the link between mitochondria and diseases with distinct anatomical and cellular patterns, how mitochondrial composition varies across brain regions and cell types remains poorly explored. Here, we analyze mitochondrial composition in different brain regions and cell types in adult mice, showing composition differs by region and cell lineage. Our work provides a resource of genes enriched in certain cell types or regions that improves our understanding of how mitochondrial composition influences brain function in health and disease.
    Keywords:  GABA; Glutamate; Mitochondria; Mitochondrial Ribosome; Respiratory Chain; Solute transporter
    DOI:  https://doi.org/10.1523/ENEURO.0232-21.2021
  9. Front Microbiol. 2021 ;12 686049
      BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.
    Keywords:  BipA; conditional protein expression; large subunit maturation; ribosome biogenesis; stress response; suboptimal temperature growth
    DOI:  https://doi.org/10.3389/fmicb.2021.686049
  10. Methods Enzymol. 2021 ;pii: S0076-6879(21)00184-1. [Epub ahead of print]656 495-519
      With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNAfMet. By mis-acylating tRNAfMet, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNAfMet, prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers.
    Keywords:  Chemical biology; Genetic code expansion; Genetically encoded materials; Non-canonical amino acids; Synthetic biology; Translation initiation
    DOI:  https://doi.org/10.1016/bs.mie.2021.05.002