Affordable Access

Publisher Website

In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g.

Authors
  • Abaeva, Irina S1
  • Arhab, Yani1
  • Miścicka, Anna1
  • Hellen, Christopher U T1
  • Pestova, Tatyana V2
  • 1 Department of Cell Biology, State University of New York Downstate Health Sciences University, Brooklyn, New York 11203, USA.
  • 2 Department of Cell Biology, State University of New York Downstate Health Sciences University, Brooklyn, New York 11203, USA [email protected].
Type
Published Article
Journal
Genes & development
Publication Date
Sep 01, 2023
Volume
37
Issue
17-18
Pages
844–860
Identifiers
DOI: 10.1101/gad.350829.123
PMID: 37821106
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on β-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment. © 2023 Abaeva et al.; Published by Cold Spring Harbor Laboratory Press.

Report this publication

Statistics

Seen <100 times