Structural insights into the mammalian late-stage initiation complexes

Simonetti, Angelita; Guca, Ewelina; Bochler, Anthony; Kuhn, Lauriane; Hashem, Yaser

doi:10.1101/779504

Cited by 13 publications

(43 citation statements)

References 95 publications

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“…Consistent with the archaeal case, the tails of uS13 and uS19 were recently observed in contact with the anticodon stem of the initiator tRNA in a mammalian late-stage initiation complex ( Simonetti et al, 2020 ). On another hand, uS13 and uS19 are also involved in translation elongation, as observed recently in a mammalian elongation complex ( Bhaskar et al, 2020 ) and during the translocation step in bacteria ( Zhou et al, 2013 ).…”

Section: Three Universal Proteins Participate In the Selection Of Thesupporting

confidence: 69%

“…Notable differences are, however, observed. First, in the yeast ribosome, eS17 has a long C-terminal extension contacting the mRNA and second, eS26 stabilizes the 5′ end of the mRNA ( Llacer et al, 2018 ; Simonetti et al, 2020 ). Interestingly, eS26 was proposed to be involved in recognition of Kozak sequence elements ( Ferretti et al, 2017 ).…”

Section: The Sd Duplex Is Bound In An Mrna Exit Channel That Differs mentioning

confidence: 99%

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Recent Advances in Archaeal Translation Initiation

et al. 2020

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Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genomewide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed.

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Section: Three Universal Proteins Participate In the Selection Of Thesupporting

confidence: 69%

Section: The Sd Duplex Is Bound In An Mrna Exit Channel That Differs mentioning

confidence: 99%

Recent Advances in Archaeal Translation Initiation

et al. 2020

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“…For general interpretation of 43S PIC and non-translating 80S, the refined models of the 40S head and body determined for the in vitro 40S-Nsp1 complex were docked as rigid bodies in UCSF Chimera 31 . To highlight the regions of the initiation factors in the cryo-EM map, initiation factors IF2 and IF3 were taken from PDB 6YAM 36 and docked similarly. A homology model for the missing eIF2β subunit was obtained using PHYRE2 (ref.…”

Section: Transient Expression Of Flag-nsp1 In Hela Cells and Puromycimentioning

confidence: 99%

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation

Schubert

Karousis

Jomaa

et al. 2020

Nat Struct Mol Biol

507

417

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ARS-CoV-2 is the causing agent of the COVID-19 pandemic and belongs to the genus of beta-coronaviruses, with enveloped, positive sense and single-stranded genomic RNA 1. On entering host cells, the viral genomic RNA is translated by the cellular protein synthesis machinery to produce a set of non-structural proteins (NSPs) 2. NSPs render the cellular conditions favorable for viral infection and viral mRNA synthesis 3. Coronaviruses have evolved specialized mechanisms to hijack the host gene expression machinery and employ cellular resources to regulate viral protein production. Such mechanisms are common for many viruses and include inhibition of host protein synthesis and endonucleolytic cleavage of host messenger RNAs (mRNAs) 4,5. In cells infected with the closely related SARS-CoV, one of the most enigmatic viral proteins is the host shutoff factor, Nsp1. Nsp1 is encoded by the gene closest to the 5′ end of the viral genome and is among the first proteins to be expressed after cell entry and infection to repress multiple steps of host protein expression 6-9. Initial structural characterization of the isolated SARS-CoV Nsp1 protein revealed the structure of its N-terminal domain, whereas its C-terminal region was flexibly disordered 10. Furthermore, it was shown that SARS-CoV Nsp1 suppresses host innate immune functions, mainly by targeting type I interferon expression and antiviral signaling pathways 11. Taken together, Nsp1 serves as a potential virulence factor for coronaviruses and represents an attractive target for live attenuated vaccine development 12,13. To provide molecular insights into the mechanism of SARS-CoV-2 Nsp1-mediated translation inhibition, we solved the structures of ribosomal complexes isolated from HEK293 lysates supplemented with recombinant purified Nsp1 as well as of an in vitro reconstituted 40S-Nsp1 complex using cryo-EM. We complement our findings by reporting in vitro and in vivo translation inhibition in the presence of Nsp1 that is relieved after mutating key interacting residues. Furthermore, we show that the translation output of reporters containing full-length viral 5′ untranslated regions (UTRs) is significantly enhanced, which could explain how Nsp1 inhibits global translation while still translating sufficient amounts of viral mRNAs. Results C-terminal domain of SARS-CoV-2 Nsp1 binds to the mRNA entry channel. To elucidate the mechanism of how Nsp1 inhibits translation, we aimed to identify the structures of potential ribosomal complexes as binding targets (Fig. 1). Previously, it has been suggested that Nsp1 mainly targets the ribosome at the translation initiation step 9. We thus treated lysed HEK293E cells with bacterially expressed and purified Nsp1 and loaded the cleared lysate on a sucrose gradient. Fractions containing ribosomal particles were then analyzed for the presence of Nsp1. Interestingly, Nsp1 not only co-migrated with 40S particles, but also with 80S ribosomal complexes (Fig. 1c), suggesting that it interacts with a range of different ribosomal states. W...

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“…The region of homology with SERBP1 is shown in green. The observed NSP1 binding sites on the 18S rRNA are demarcated in cyan on the structure of the human 40S ribosome (PDB: 6G5H ; gray )( Ameismeier et al., 2018 ), relative to the mRNA path (purple; 6YAL)( Simonetti et al., 2020 ), and known clogging factors (E) SERBP1 (green; 6MTE)( Brown et al., 2018 ) and (F) Stm1 (orange; 4V88)( Ben-Shem et al., 2011 ). (D) An mRNA encoding GFP was added to HeLa cell extracts along with different concentrations of purified NSP1 protein (x axis).…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

Banerjee

Blanco

Bruce

et al. 2020

Cell

521

649

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

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Structural insights into the mammalian late-stage initiation complexes

Cited by 13 publications

References 95 publications

Recent Advances in Archaeal Translation Initiation

Recent Advances in Archaeal Translation Initiation

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

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