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.

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

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.

Tethered Function Assays to Elucidate the Role of RNA-Binding Proteins.

The fate of each RNA molecule is strongly determined by RNA-binding proteins (RBPs) which accompany transcripts from its synthesis to its degradation. To elucidate the effect of a specific RBP on bound RNA, it can be artificially recruited to a specific site on a reporter mRNA that can be followed by a variety of methods. In this so-called tethering assay, the protein of interest (POI) is fused to the coat protein of the MS2 bacteriophage and expressed in your favorite cells together with a reporter gene containing MS2 binding sites. The MS2 binding sites are recognized by the MS2 coat protein (MS2CP) with high affinity and specificity and by doing so, the POI is tethered to the reporter RNA. Here, we describe how with the help of this assay the human cytoplasmic poly(A) binding protein is recruited to a mini-µ RNA reporter, thereby influencing the stability of the reporter transcript.

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.

40S hnRNP particles are a novel class of nuclear biomolecular condensates.

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.

A comprehensive coverage insurance for cells: revealing links between ribosome collisions, stress responses and mRNA surveillance.

Cells of metazoans respond to internal and external stressors by activating stress response pathways that aim for re-establishing cellular homoeostasis or, if this cannot be achieved, triggering programmed cell death. Problems during translation, arising from defective mRNAs, tRNAs, ribosomes or protein misfolding, can activate stress response pathways as well as mRNA surveillance and ribosome quality control programs. Recently, ribosome collisions have emerged as a central signal for translational stress and shown to elicit different stress responses. Here, we review our current knowledge about the intricate mutual connections between ribosome collisions, stress response pathways and mRNA surveillance. A central factor connecting the sensing of collided ribosomes with degradation of the nascent polypeptides, dissociation of the stalled ribosomes and degradation of the mRNA by no-go or non-stop decay is the E3-ligase ZNF598. We tested whether ZNF598 also plays a role in nonsense-mediated mRNA decay (NMD) but found that it is dispensable for this translation termination-associated mRNA surveillance pathway, which in combination with other recent data argues against stable ribosome stalling at termination codons being the NMD-triggering signal.

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.

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.

Translation mediated by the nuclear cap-binding complex is confined to the perinuclear region via a CTIF-DDX19B interaction.

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Characterisation of the Semliki Forest Virus-host cell interactome reveals the viral capsid protein as an inhibitor of nonsense-mediated mRNA decay.

The positive-sense, single-stranded RNA alphaviruses pose a potential epidemic threat. Understanding the complex interactions between the viral and the host cell proteins is crucial for elucidating the mechanisms underlying successful virus replication strategies and for developing specific antiviral interventions. Here we present the first comprehensive protein-protein interaction map between the proteins of Semliki Forest Virus (SFV), a mosquito-borne member of the alphaviruses, and host cell proteins. Among the many identified cellular interactors of SFV proteins, the enrichment of factors involved in translation and nonsense-mediated mRNA decay (NMD) was striking, reflecting the virus' hijacking of the translation machinery and indicating viral countermeasures for escaping NMD by inhibiting NMD at later time points during the infectious cycle. In addition to observing a general inhibition of NMD about 4 hours post infection, we also demonstrate that transient expression of the SFV capsid protein is sufficient to inhibit NMD in cells, suggesting that the massive production of capsid protein during the SFV reproduction cycle is responsible for NMD inhibition.

FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

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.

Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay.

To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3' UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3' UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3' UTR, where it can resume translation in-frame as well as out-of-frame.

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.

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.

FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention.

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.