Cap-independent translation and a precisely localized RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response
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ABSTRACT:
PROVIDER: PRJNA797428 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17622353_1.fastq.gz | Fastqsanger.gz | |||
| SRR17622353_2.fastq.gz | Fastqsanger.gz | |||
| SRR17622354_1.fastq.gz | Fastqsanger.gz | |||
| SRR17622354_2.fastq.gz | Fastqsanger.gz | |||
| SRR17622355_1.fastq.gz | Fastqsanger.gz |
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Project description:Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represseses translation and induces mRNA degradation, while the host elicits anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-meidated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5’UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5’UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5’UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets
2022-01-17 | GSE193732 | GEO
Project description:Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5'UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5'UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5'UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.
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Project description:To investigate gene specificity at the level of translation in both the human genome and viruses we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncover thousands of novel cap-independent translation sequences and provide insights on the landscape of translational regulation in both human and viruses. We find extensive translational elements in the 3â untranslated region (3âUTR) of human transcripts and the polyprotein region of un-capped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity we identify potential mechanisms of secondary structure, short sequence motif and base-pairing with the 18S rRNA. Furthermore, we systematically map the 18S rRNA regions for which reverse complementary enhance translation. Thus we provide insights into the mechanisms of translational control in humans and viruses. high-throughput bicistronic assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).
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Project description:To investigate gene specificity at the level of translation in both the human genome and viruses, we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncovered thousands of novel cap-independent translation sequences, and we provide insights on the landscape of translational regulation in both humans and viruses. We find extensive translational elements in the 3′ untranslated region of human transcripts and the polyprotein region of uncapped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity, we identify potential mechanisms of secondary structure, short sequence motif,and base pairing with the 18S ribosomal RNA (rRNA). Furthermore, we systematically map the 18S rRNA regions for which reverse complementarity enhances translation. Thus, we make available insights into the mechanisms of translational control in humans and viruses.
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Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We present eIF3a PAR-iCLIP data which demonstrate that eIF3 predominantly binds mRNAs within the 5'UTR. eIF3 binding sites are also in proximity to m6A residues within the 5'UTR of cellular mRNAs. Two replicates of eIF3a PAR-iCLIP in HEK293T cells.
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Project description:Plitidepsin is an antitumoral drug safe for treating COVID-19 infection that impairs SARS-CoV-2 replication by targeting the translation elongation factor eEF1A. Here we show that plitidepsin decreased all SARS-CoV-2 transcripts along the early translation of viral R1AB polyproteins and the late synthesis of structural viral proteins. It also reduced de novo cap-dependent translation of viral and non-viral RNAs but affected less than 13% of the host proteome, thus preserving cellular viability. In response to plitidepsin, cells upregulated EIF2AK3 and additional proteins that reduce translation. Other proteins supported proteostasis via ribosome synthesis and cap-independent translation routes, which relied on eIF4G2, PABPC1, and readers such as IGF2BP2. While cap- or internal ribosome entry sites (IRES)-mediated translation pathways were inhibited by plitidepsin, impact on N6-methyladenosine (m6A) translation was limited. The molecular landscape reprogrammed by cells treated with plitidepsin predicted a potential broad-spectrum activity against viruses translated via cap- or IRES-dependent pathways that were suppressed by m6A. In agreement, plitidepsin inhibited the replication of members from the Coronaviridae, Flaviviridae, Pneumoviridae and Herpesviridae families. Yet, it failed to block retroviruses that exploit m6A synthesis routes and are inhibited by drugs against specific m6A readers such as IGF2BP2. By deciphering the molecular fingerprint of cells treated with host-directed therapies targeting protein translation, we identified a rational approach to select for broad-spectrum antivirals with complementary activities and potential to counteract future pandemic viruses.
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Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We performed m6A profiling in cells subjected to heat shock stress and observed increased 5'UTR methylation after heat shock. For these studies, we used single-nucleotide resolution m6A mapping (miCLIP), which enables the detection of m6A residues separately from m6Am residues. Thus, the goal of these experiments was to identify m6A residues which are increased in the 5'UTR after heat shock stress.
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