Biology subjects

Hellen, C. U. T.

Publications and source records attributed to Hellen, C. U. T..

3 recordsLinked to original sources

bioRxiv2023

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

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 {beta}-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 eIF3gs RRM domain. The cleavage site was located in the coding region 18 nucleotides 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 Nsp1s N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3gs RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3gs RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

molecular biology↗

bioRxiv2021

Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

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). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 [A] that cover all major stages from 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 for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

molecular biology↗

bioRxiv2020

The Halastavi arva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi arva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

molecular biology↗