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...

AbstractThe non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.

Besides degrading aberrant mRNAs that harbor a premature translation termination codon (PTC), nonsense-mediated mRNA decay (NMD) also targets many seemingly "normal" mRNAs that encode for full-length proteins. To identify a bona fide set of such endogenous NMD targets in human cells, we applied a meta-analysis approach in which we combined transcriptome profiling of knockdowns and rescues of the three NMD factors UPF1, SMG6, and SMG7. We provide evidence that this combinatorial approach identifies NMD-targeted transcripts more reliably than previous attempts that focused on inactivation of single NMD factors. Our data revealed that SMG6 and SMG7 act on essentially the same transcripts, indicating extensive redundancy between the endo-and exonucleolytic decay routes. Besides mRNAs, we also identified as NMD targets many long noncoding RNAs as well as miRNA and snoRNA host genes. The NMD target feature with the most predictive value is an intron in the 3 ′ UTR, followed by the presence of upstream open reading frames (uORFs) and long 3 ′ UTRs. Furthermore, the 3 ′ UTRs of NMD-targeted transcripts tend to have an increased GC content and to be phylogenetically less conserved when compared to 3 ′ UTRs of NMD insensitive transcripts.

Nonsense-mediated mRNA decay (NMD) was originally coined to define a quality control mechanism that targets mRNAs with truncated open reading frames due to the presence of a premature termination codon. Meanwhile, it became clear that NMD has a much broader impact on gene expression and additional biological functions beyond quality control are continuously being discovered. We review here the current views regarding the molecular mechanisms of NMD, according to which NMD ensues on mRNAs that fail to terminate translation properly, and point out the gaps in our understanding. We further summarize the recent literature on an ever-rising spectrum of biological processes in which NMD appears to be involved, including homeostatic control of gene expression, development and differentiation, as well as viral defense.

Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messenger RNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecular mechanism of target RNA selection remain unsolved. Besides degrading defective mRNAs harboring premature termination codons (PTCs), NMD also targets many mRNAs encoding functional full-length proteins. Thus, NMD impacts on a cell's transcriptome and is implicated in a range of biological processes that affect a broad spectrum of cellular homeostasis. Here, we focus on the steps involved in the recognition of NMD targets and the activation of NMD. We summarize the accumulating evidence that tightly links NMD to translation termination and we further discuss the recruitment and activation of the mRNA degradation machinery and the regulation of this complex series of events. Finally, we review emerging ideas concerning the mechanistic details of NMD activation and the potential role of NMD as a general surveyor of translation efficacy.

In the version of this article initially published online, in Table 1, the PDB accession code for the S20G 2PF cryo-EM structure was incorrect. The correct accession code is PDB 6ZRQ. The error has been corrected in the print, PDF and HTML versions of the article.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

Background Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. Results To identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. Conclusions Long-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.