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  1. Article: mRNA Targeting, Transport and Local Translation in Eukaryotic Cells: From the Classical View to a Diversity of New Concepts.

    Lashkevich, Kseniya A / Dmitriev, Sergey E

    Molecular biology

    2021  Volume 55, Issue 4, Page(s) 507–537

    Abstract: Spatial organization of protein biosynthesis in the eukaryotic cell has been studied for more than fifty years, thus many facts have already been included in textbooks. According to the classical view, mRNA transcripts encoding secreted and transmembrane ...

    Abstract Spatial organization of protein biosynthesis in the eukaryotic cell has been studied for more than fifty years, thus many facts have already been included in textbooks. According to the classical view, mRNA transcripts encoding secreted and transmembrane proteins are translated by ribosomes associated with endoplasmic reticulum membranes, while soluble cytoplasmic proteins are synthesized on free polysomes. However, in the last few years, new data has emerged, revealing selective translation of mRNA on mitochondria and plastids, in proximity to peroxisomes and endosomes, in various granules and at the cytoskeleton (actin network, vimentin intermediate filaments, microtubules and centrosomes). There are also long-standing debates about the possibility of protein synthesis in the nucleus. Localized translation can be determined by targeting signals in the synthesized protein, nucleotide sequences in the mRNA itself, or both. With RNA-binding proteins, many transcripts can be assembled into specific RNA condensates and form RNP particles, which may be transported by molecular motors to the sites of active translation, form granules and provoke liquid-liquid phase separation in the cytoplasm, both under normal conditions and during cell stress. The translation of some mRNAs occurs in specialized "translation factories," assemblysomes, transperons and other structures necessary for the correct folding of proteins, interaction with functional partners and formation of oligomeric complexes. Intracellular localization of mRNA has a significant impact on the efficiency of its translation and presumably determines its response to cellular stress. Compartmentalization of mRNAs and the translation machinery also plays an important role in viral infections. Many viruses provoke the formation of specific intracellular structures, virus factories, for the production of their proteins. Here we review the current concepts of the molecular mechanisms of transport, selective localization and local translation of cellular and viral mRNAs, their effects on protein targeting and topogenesis, and on the regulation of protein biosynthesis in different compartments of the eukaryotic cell. Special attention is paid to new systems biology approaches, providing new cues to the study of localized translation.
    Language English
    Publishing date 2021-05-30
    Publishing country Russia (Federation)
    Document type Journal Article
    ZDB-ID 213541-3
    ISSN 1608-3245 ; 0026-8933
    ISSN (online) 1608-3245
    ISSN 0026-8933
    DOI 10.1134/S0026893321030080
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Monitoring integrated stress response in live

    Lidsky, Peter V / Yuan, Jing / Lashkevich, Kseniya A / Dmitriev, Sergey E / Andino, Raul

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, ... ...

    Abstract Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, our understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder our ability to track and manipulate the ISR in vivo. To overcome these limitations, we have successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables us to label Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, we have unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of our reporter, we have engineered ISR-null eIF2αS50A mutant flies for stress response analysis. Our results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.
    Language English
    Publishing date 2023-07-14
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.07.13.548942
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: A Solitary Stalled 80S Ribosome Prevents mRNA Recruitment to Stress Granules.

    Fedorovskiy, Artem G / Burakov, Anton V / Terenin, Ilya M / Bykov, Dmitry A / Lashkevich, Kseniya A / Popenko, Vladimir I / Makarova, Nadezhda E / Sorokin, Ivan I / Sukhinina, Anastasia P / Prassolov, Vladimir S / Ivanov, Pavel V / Dmitriev, Sergey E

    Biochemistry. Biokhimiia

    2023  Volume 88, Issue 11, Page(s) 1786–1799

    Abstract: In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called ...

    Abstract In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.
    MeSH term(s) RNA, Messenger/metabolism ; Protein Biosynthesis ; Stress Granules ; Cytoplasmic Granules ; Ribosomes/metabolism ; Protein Synthesis Inhibitors/pharmacology ; RNA-Binding Proteins/metabolism
    Chemical Substances RNA, Messenger ; Protein Synthesis Inhibitors ; RNA-Binding Proteins
    Language English
    Publishing date 2023-12-18
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1109-5
    ISSN 1608-3040 ; 0006-2979 ; 0320-9717
    ISSN (online) 1608-3040
    ISSN 0006-2979 ; 0320-9717
    DOI 10.1134/S000629792311010X
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.

    Lashkevich, Kseniya A / Shlyk, Valeriya I / Kushchenko, Artem S / Gladyshev, Vadim N / Alkalaeva, Elena Z / Dmitriev, Sergey E

    Biomolecules

    2020  Volume 10, Issue 6

    Abstract: Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in ... ...

    Abstract Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.
    MeSH term(s) Biological Assay/methods ; Cell-Free System/physiology ; Codon, Nonsense ; Codon, Terminator/genetics ; DNA Mutational Analysis ; Genes, Reporter ; Humans ; In Vitro Techniques ; Luciferases/genetics ; Luciferases/metabolism ; Mutation Rate ; Peptide Chain Termination, Translational/genetics ; Peptide Chain Termination, Translational/physiology ; Peptide Termination Factors/genetics ; Peptide Termination Factors/metabolism ; Protein Biosynthesis/genetics
    Chemical Substances Codon, Nonsense ; Codon, Terminator ; ETF1 protein, human ; Peptide Termination Factors ; Luciferases (EC 1.13.12.-)
    Language English
    Publishing date 2020-06-16
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2701262-1
    ISSN 2218-273X ; 2218-273X
    ISSN (online) 2218-273X
    ISSN 2218-273X
    DOI 10.3390/biom10060911
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

    Osterman, Ilya A / Wieland, Maximiliane / Maviza, Tinashe P / Lashkevich, Kseniya A / Lukianov, Dmitrii A / Komarova, Ekaterina S / Zakalyukina, Yuliya V / Buschauer, Robert / Shiriaev, Dmitrii I / Leyn, Semen A / Zlamal, Jaime E / Biryukov, Mikhail V / Skvortsov, Dmitry A / Tashlitsky, Vadim N / Polshakov, Vladimir I / Cheng, Jingdong / Polikanov, Yury S / Bogdanov, Alexey A / Osterman, Andrei L /
    Dmitriev, Sergey E / Beckmann, Roland / Dontsova, Olga A / Wilson, Daniel N / Sergiev, Petr V

    Nature chemical biology

    2020  Volume 16, Issue 10, Page(s) 1071–1077

    Abstract: The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we ... ...

    Abstract The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.
    MeSH term(s) Amycolatopsis/drug effects ; Amycolatopsis/genetics ; Amycolatopsis/metabolism ; Binding Sites ; Cryoelectron Microscopy ; Drug Resistance, Bacterial ; Escherichia coli ; Gene Expression Regulation, Bacterial/drug effects ; HEK293 Cells ; Humans ; Microbial Sensitivity Tests ; Models, Molecular ; Mutation ; Naphthacenes/chemistry ; Naphthacenes/pharmacology ; Protein Binding ; Protein Biosynthesis/drug effects ; Protein Conformation ; Ribosomes/metabolism
    Chemical Substances Naphthacenes ; tetracenomycin X (121245-07-6)
    Language English
    Publishing date 2020-06-29
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2202962-X
    ISSN 1552-4469 ; 1552-4450
    ISSN (online) 1552-4469
    ISSN 1552-4450
    DOI 10.1038/s41589-020-0578-x
    Database MEDical Literature Analysis and Retrieval System OnLINE

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