Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 NSP1 protein
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ABSTRACT:
PROVIDER: PRJNA665035 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12693500.fastq.gz | Fastqsanger.gz | |||
| SRR12693501.fastq.gz | Fastqsanger.gz | |||
| SRR12693502.fastq.gz | Fastqsanger.gz | |||
| SRR12693503.fastq.gz | Fastqsanger.gz | |||
| SRR12693504.fastq.gz | Fastqsanger.gz |
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Project description:Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, NSP1, for shutting down host translation. Despite this, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing NSP1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes preferentially translated in the context of NSP1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we also uncover potential mechanisms of preferential translation through the presence of shared sites for specific RNA binding proteins and a remarkable enrichment for 5′ terminal oligo-pyrimidine tracts. Collectively, the present study suggests fine tuning of host gene expression and translation by NSP1 despite its global repressive effect on host protein synthesis.
2020-10-01 | GSE158374 | GEO
Project description:Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.
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Project description:Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of NSP1. Finally, we found that LARP1, a key effector protein in the mTOR pathway may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.
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2011-11-01 | E-GEOD-26548 | biostudies-arrayexpress
Project description:In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. However, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains unclear. As the conserved N-terminal domain (NTD) and linker region has been implicated with this process, we set out to characterize the interactome of Nsp1 and its topology by crosslinking mass spectrometry. The distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit. We find that Nsp1 is in contact with 40S proteins lining the mRNA entry channel and the G subunit of the eIF3 complex. We further show that Nsp1 predominantly binds to initiating ribosomes by affinity purification mass spectrometry and sucrose gradient ultracentrifugation. Our results point towards a potential mechanism by which Nsp1 is preferentially recruited to host-translating ribosomes, leading to selective inhibition of canonical initiation intermediates and subsequent mRNA degradation.
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Project description:In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. However, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains unclear. As the conserved N-terminal domain (NTD) and linker region has been implicated with this process, we set out to characterize the interactome of Nsp1 and its topology by crosslinking mass spectrometry. The distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit. We find that Nsp1 is in contact with 40S proteins lining the mRNA entry channel and the G subunit of the eIF3 complex. We further show that Nsp1 predominantly binds to initiating ribosomes by affinity purification mass spectrometry and sucrose gradient ultracentrifugation. Our results point towards a potential mechanism by which Nsp1 is preferentially recruited to host-translating ribosomes, leading to selective inhibition of canonical initiation intermediates and subsequent mRNA degradation.
2023-02-15 | PXD031688 | JPOST Repository
Project description:Legume GRAS-type transcription factors NSP1 and NSP2 are essential for Rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression upon symbiotic signalling. However, legume NSP1 and NSP2 can be functionally replaced by non-legume orthologs; including rice (Oryza sativa) OsNSP1 and OsNSP2. This shows that both proteins are functionally conserved in higher plants, suggesting an ancient function that was conserved during evolution. Here we show that NSP1 and NSP2 are indispensable for strigolactone biosynthesis in the legume Medicago truncatula as well as rice. Mutant nsp1-nsp2 plants hardly produce strigolactones. The lack of strigolactone biosynthesis coincides with strongly reduced DWARF27 expression in both species. Rice and Medicago represent distinct phylogenetic lineages that split ~150 million years ago. Therefore we conclude that regulation of strigolactone biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. Since strigolactone biosynthesis is highly regulated by environmental conditions like phosphate starvation, NSP1 and NSP2 will be important tools in future studies on the molecular mechanisms by which environmental sensing is translated into regulation of strigolactone biosynthesis. As NSP1 and NSP2 are single copy genes in legumes, it implies that a single protein complex fulfills a dual regulatory function of different downstream targets; symbiotic and non-symbiotic, respectively.
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