Universal features of Nsp1-mediated translational shutdown by coronaviruses.

Nonstructural protein 1 (Nsp1) produced by coronaviruses inhibits host protein synthesis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp1 C-terminal domain was shown to bind the ribosomal mRNA channel to inhibit translation, but it is unclear whether this mechanism is broadly used by coronaviruses, whether the Nsp1 N-terminal domain binds the ribosome, or how Nsp1 allows viral RNAs to be translated. Here, we investigated Nsp1 from SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), and Bat-Hp-CoV coronaviruses using structural, biophysical, and biochemical experiments, revealing a conserved role for the C-terminal domain. Additionally, the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A accommodation. Structure-based experiments demonstrated the importance of decoding center interactions in all three coronaviruses and showed that the same regions of Nsp1 are necessary for the selective translation of viral RNAs. Our results provide a mechanistic framework to understand how Nsp1 controls preferential translation of viral RNAs.

Coronavirus takeover of host cell translation and intracellular antiviral response: a molecular perspective.

Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion.

The broader sense of nonsense.

The term 'nonsense-mediated mRNA decay' (NMD) was initially coined to describe the translation-dependent degradation of mRNAs harboring premature termination codons (PTCs), but it is meanwhile known that NMD also targets many canonical mRNAs with numerous biological implications. The molecular mechanisms determining on which RNAs NMD ensues are only partially understood. Considering the broad range of NMD-sensitive RNAs and the variable degrees of their degradation, we highlight here the hallmarks of mammalian NMD and point out open questions. We review the links between NMD and disease by summarizing the role of NMD in cancer, neurodegeneration, and viral infections. Finally, we describe strategies to modulate NMD activity and specificity as potential therapeutic approaches for various diseases.

The art of hijacking: how Nsp1 impacts host gene expression during coronaviral infections.

Non-structural protein 1 (Nsp1) is one of the first proteins produced during coronaviral infections. It plays a pivotal role in hijacking and rendering the host gene expression under the service of the virus. With a focus on SARS-CoV-2, this review presents how Nsp1 selectively inhibits host protein synthesis and induces mRNA degradation of host but not viral mRNAs and blocks nuclear mRNA export. The clinical implications of this protein are highlighted by showcasing the pathogenic role of Nsp1 through the repression of interferon expression pathways and the features of viral variants with mutations in the Nsp1 coding sequence. The ability of SARS-CoV-2 Nsp1 to hinder host immune responses at an early step, the absence of homology to any human proteins, and the availability of structural information render this viral protein an ideal drug target with therapeutic potential.

Production of human translation-competent lysates using dual centrifugation.

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways.

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.

Universal features of Nsp1-mediated translational shutdown by coronaviruses.

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells.

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.

Human NMD ensues independently of stable ribosome stalling.

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.

Author Correction: SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. We observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in 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 global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.

Nonsense-Mediated mRNA Decay Begins Where Translation Ends.

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.

Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact.

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. WIREs RNA 2016, 7:661-682. doi: 10.1002/wrna.1357 For further resources related to this article, please visit the WIREs website.

A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.