bims-tricox Biomed News
on Translation, ribosomes and COX
Issue of 2022–07–17
eleven papers selected by
Yash Verma, University of Delhi South Campus
  1. Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.
  2. The functional role of the eukaryote-specific motif YxxPKxYxK of the human ribosomal protein eS26 in translation.
  3. Editorial: mRNA Translational Control as a Mechanism of Post-transcriptional Gene Regulation.
  4. DHX37 and 46,XY DSD: A New Ribosomopathy?
  5. Three-dimensional structure-guided evolution of a ribosome with tethered subunits.
  6. Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM.
  7. Cryo-EM structure of a monomeric yeast S. cerevisiae complex IV isolated with maltosides: Implications in supercomplex formation.
  8. Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.
  9. Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells.
  10. Characterizing and Improving pET Vectors for Cell-free Expression.
  11. The nuclear encoded Cox7c mRNA co-transport with mitochondria along axons via coding-region dependent mechanism.
  1. EMBO J. 2022 Jul 13. e110581
    Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.
    Zuben P Brown, Irina S Abaeva, Swastik De, Christopher U T Hellen, Tatyana V Pestova, Joachim Frank.
      Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.
    Keywords:  eIF2; eIF5B; hepatitis C virus IRES; ribosome; translation initiation
    DOI:  https://doi.org/10.15252/embj.2022110581
  2. Biochim Biophys Acta Gene Regul Mech. 2022 Jul 08. pii: S1874-9399(22)00057-8. [Epub ahead of print]1865(6): 194842
    The functional role of the eukaryote-specific motif YxxPKxYxK of the human ribosomal protein eS26 in translation.
    Konstantin N Bulygin, Alexey A Malygin, Dmitri M Graifer, Galina G Karpova.
      The protein eS26 is a structural component of the eukaryotic small ribosomal subunit involved in the formation of the mRNA binding channel in the region of the exit site. By applying site-directed cross-linking to mammalian 80S ribosomes, it has been shown that the same mRNA nucleotide residues are implicated in the interaction with both eS26 and translation initiation factor 3 (eIF3) and that contacts of the protein with mRNAs are mediated by its eukaryote-specific motif YxxPKxYxK. To examine the role of eS26 in translation, we transfected HEK293T cells with plasmid constructs encoding the wild-type FLAG-labeled protein (wt-eS26FLAG) or its forms with either a single substitution of any conserved amino acid residue in the above motif, or a simultaneous replacement of all the five ones (5A). The western blot analysis of fractions of polysome profiles from the transfected cells revealed no effects of the single mutations in eS26, but showed that the replacement of the five conserved residues led to the increased share of the light polysome fraction compared to that detected with control, wt-eS26FLAG-producing cells. In addition, the above fraction exhibited the enhanced content of the eIF3e subunit that is known to promote selective translation. These findings, together with real-time PCR data on the relative contents of specific mRNAs in light and heavy polysomes from cells producing the mutant 5A compared to those from control cells, suggest a possible involvement of the YxxPKxYxK motif of eS26 in the fine regulation of translation to maintain the required balance of synthesized proteins.
    Keywords:  Amino acid substitutions in human ribosomal protein eS26; Initiation factor eIF3; Polysome profiling; Role of eS26 in the regulation of translation of specific mRNAs; Transient transfection of HEK293T cells
    DOI:  https://doi.org/10.1016/j.bbagrm.2022.194842
  3. Front Mol Biosci. 2022 ;9 947516
    Editorial: mRNA Translational Control as a Mechanism of Post-transcriptional Gene Regulation.
    Daniel L Kiss, Deepika Vasudevan, C Kiong Ho, Neva Caliskan.
      
    Keywords:  RNA binding proteins; bacterial translation; mRNA translational control; noncanonical translation factors; ribosome frameshifting; ribosome profiling; translation initiation factors; untranslated regions (UTRs)
    DOI:  https://doi.org/10.3389/fmolb.2022.947516
  4. Sex Dev. 2022 Jul 14. 1-13
    DHX37 and 46,XY DSD: A New Ribosomopathy?
    Kenneth McElreavey, Eric Pailhoux, Anu Bashamboo.
      Recently, a series of recurrent missense variants in the RNA-helicase DHX37 have been reported associated with either 46,XY gonadal dysgenesis, 46,XY testicular regression syndrome (TRS), or anorchia. All affected children have non-syndromic forms of disorders/differences of sex development (DSD). These variants, which involve highly conserved amino acids within known functional domains of the protein, are predicted by in silico tools to have a deleterious effect on helicase function. DHX37 is required for ribosome biogenesis in eukaryotes, and how these variants cause DSD is unclear. The relationship between DHX37 and human congenital disorders is complex as compound heterozygous as well as de novo heterozygous missense variants in DHX37 are also associated with a complex congenital developmental syndrome (NEDBAVC, neurodevelopmental disorder with brain anomalies and with or without vertebral or cardiac anomalies; OMIM 618731), consisting of microcephaly, global developmental delay, seizures, facial dysmorphia, and kidney and cardiac anomalies. Here, we will give a brief overview of ribosome biogenesis and the role of DHX37 in this process. We will discuss variants in DHX37, their contribution to human disease in the general context of human ribosomopathies, and the possible disease mechanisms that may be involved.
    Keywords:  DHX37; Disorders/differences of sex development; RNA helicase; Ribosome biogenesis; Ribosomopathy
    DOI:  https://doi.org/10.1159/000522004
  5. Nat Chem Biol. 2022 Jul 14.
    Three-dimensional structure-guided evolution of a ribosome with tethered subunits.
    Do Soon Kim, Andrew Watkins, Erik Bidstrup, Joongoo Lee, Ved Topkar, Camila Kofman, Kevin J Schwarz, Yan Liu, Grigore Pintilie, Emily Roney, Rhiju Das, Michael C Jewett.
      RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.
    DOI:  https://doi.org/10.1038/s41589-022-01064-w
  6. Front Mol Biosci. 2022 ;9 892459
    Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM.
    Yixin Xu, Shangyu Dang.
      Cryo-sample preparation is a vital step in the process of obtaining high-resolution structures of macromolecules by using the single-particle cryo-electron microscopy (cryo-EM) method; however, cryo-sample preparation is commonly hampered by high uncertainty and low reproducibility. Specifically, the existence of air-water interfaces during the sample vitrification process could cause protein denaturation and aggregation, complex disassembly, adoption of preferred orientations, and other serious problems affecting the protein particles, thereby making it challenging to pursue high-resolution 3D reconstruction. Therefore, sample preparation has emerged as a critical research topic, and several new methods for application at various preparation stages have been proposed to overcome the aforementioned hurdles. Here, we summarize the methods developed for enhancing the quality of cryo-samples at distinct stages of sample preparation, and we offer insights for developing future strategies based on diverse viewpoints. We anticipate that cryo-sample preparation will no longer be a limiting step in the single-particle cryo-EM field as increasing numbers of methods are developed in the near future, which will ultimately benefit the entire research community.
    Keywords:  air-water interface; grid modification; particle distribution; sample preparation; single-particle cryo-electron microscopy
    DOI:  https://doi.org/10.3389/fmolb.2022.892459
  7. Biochim Biophys Acta Bioenerg. 2022 Jul 12. pii: S0005-2728(22)00060-3. [Epub ahead of print] 148591
    Cryo-EM structure of a monomeric yeast S. cerevisiae complex IV isolated with maltosides: Implications in supercomplex formation.
    Gabriel Ing, Andrew M Hartley, Nikos Pinotsis, Amandine Maréchal.
      In mitochondria, complex IV (CIV) can be found as a monomer, a dimer or in association with other respiratory complexes. The atomic structure of the yeast S. cerevisiae CIV in a supercomplex (SC) with complex III (CIII) pointed to a region of significant conformational changes compared to the homologous mammalian CIV structures. These changes involved the matrix side domain of Cox5A at the CIII-CIV interface, and it was suggested that it could be required for SC formation. To investigate this, we solved the structure of the isolated monomeric CIV from S. cerevisiae stabilised in amphipol A8-35 at 3.9 Å using cryo-electron microscopy. Only a minor change in flexibility was seen in this Cox5A region, ruling out large CIV conformational shift for interaction with CIII and confirming the different fold of the yeast Cox5A subunit compared to mammalian homologues. Other differences in structure were the absence of two canonical subunits, Cox12 and Cox13, as well as Cox26, which is unique to the yeast CIV. Their absence is most likely due to the protein purification protocol used to isolate CIV from the III-IV SC.
    Keywords:  Amphipols; Bioenergetics; Cytochrome c oxidase; Electron transport chain; Mitochondria; Supercomplexes
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148591
  8. Front Mol Biosci. 2022 ;9 882288
    Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.
    Stephan Niebling, Katharina Veith, Benjamin Vollmer, Javier Lizarrondo, Osvaldo Burastero, Janina Schiller, Angelica Struve García, Philipp Lewe, Carolin Seuring, Susanne Witt, María García-Alai.
      Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.
    Keywords:  biophysical characterization; differential scanning fluorimetry (DSF); dynamic light scattering; electron cryo-microscopy (cryo-EM); mass photometry; membrane proteins; sample preparation
    DOI:  https://doi.org/10.3389/fmolb.2022.882288
  9. J Pharm Biomed Anal. 2022 Jul 08. pii: S0731-7085(22)00347-8. [Epub ahead of print]219 114926
    Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells.
    Riccardo Rampado, Federica Giordano, Laura Moracci, Sara Crotti, Paolo Caliceti, Marco Agostini, Francesca Taraballi.
      Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.
    Keywords:  Detergent; Extraction; Mammalian cells; Membrane proteins; Protocol
    DOI:  https://doi.org/10.1016/j.jpba.2022.114926
  10. Front Bioeng Biotechnol. 2022 ;10 895069
    Characterizing and Improving pET Vectors for Cell-free Expression.
    Kara Jew, Philip E J Smith, Byungcheol So, Jillian Kasman, Javin P Oza, Michael W Black.
      Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.
    Keywords:  cell-free; in vitro; pET30; protein synthesis; template; translation
    DOI:  https://doi.org/10.3389/fbioe.2022.895069
  11. J Cell Sci. 2022 Jul 14. pii: jcs.259436. [Epub ahead of print]
    The nuclear encoded Cox7c mRNA co-transport with mitochondria along axons via coding-region dependent mechanism.
    Bar Cohen, Topaz Altman, Adi Golani-Armon, Anca F Savulescu, Amjd Ibraheem, Musa M Mhlanga, Eran Perlson, Yoav S Arava.
      Nuclear encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the transport mechanism of those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and smFISH assay revealed that endogenous mRNA encoding Cox7c is preferentially associated with mitochondria from a neuronal cell line and within primary motor neuron axons, while other mRNAs, which do not encode mitochondrial protein are much less associated. Live cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' UTR, was the key domain for the cotransport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.
    Keywords:  Axonal transport; Cox7c; Mitochondria; mRNA localization; mRNA transport
    DOI:  https://doi.org/10.1242/jcs.259436
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