Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

The eukaryotic ribosomal protein S15/uS19 is involved in fungal development and its C-terminal tail contributes to stop codon recognition

SummaryS15/uS19 is one of the fifteen universally conserved ribosomal proteins of the small ribosomal subunit. While prokaryotic uS19 is located away from the mRNA decoding site, cross-linking studies identified eukaryotic uS19 C-terminal tail as contacting the A site on the 80S ribosome. Here, we study the effects of uS19 mutations isolated as translation accuracy mutations in the filamentous fungus Podospora anserina. All mutations alter residues of uS19 C-terminal tail, and cluster to the eukaryote-specific decapeptide 138-PGIGATHSSR-147. All mutations modify fungal development and cytosolic translation, albeit differently. Two mutations (P138S and S145F) increase fungus longevity and display mild effects on translation, while others (G139D and G139C) decrease longevity, have stronger effects on translation and confer hypersensitivity to paromomycin. By mimicking P. anserina mutations in the yeast Saccharomyces cerevisiae RPS15 gene, we further show that P138S and S145F induce hyperaccurate recognition of the three stop codons, whereas G139D and G139C impair UAG and UAA codon recognition. Noteworthy, in P. anserina, uS19 genetically interacts with the eRF1 and eRF3 release factors. All together, our data indicate that uS19 C-terminal tail contributes in vivo to eukaryotic translation termination, and identify key amino acids of uS19 that potentially modulate eRF1-eRF3 interaction in the pre-termination complex.Graphical abstractAbbreviated SummaryS15/uS19 is a conserved small ribosomal protein that in eukaryotes harbors a flexible C-terminal extension proposed to interact with the A site mRNA codon during translation. Here, we describe how C-terminal variants variously affect Podospora anserina development and longevity and impact fungal ribosome and polysome formation. We reveal that stop codon recognition is significantly altered by the presence of those C-terminal variants, which either expand or on the contrary restrict termination ambiguity.

Deconstructing the individual steps of vertebrate translation initiation

Translation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control 1. Despite this centrality the series of steps involved in this process are poorly understood 2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5’ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

Conformational control of translation termination on the 70S ribosome

Translation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate translation termination. If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codon-recognition domain is fully engaged in stop-codon recognition in the decoding center. RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.