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Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed –1 ribosomal frameshifting

ABSTRACT: Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed –1 ribosomal frameshifting

PROVIDER: PRJNA927297 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23218841.fastq.gz	Fastqsanger.gz
	SRR23218842.fastq.gz	Fastqsanger.gz
	SRR23218843.fastq.gz	Fastqsanger.gz
	SRR23218844.fastq.gz	Fastqsanger.gz
	SRR23218845.fastq.gz	Fastqsanger.gz

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Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed –1 ribosomal frameshifting

Project description:Many positive-strand RNA viruses, including all known coronaviruses, employ programmed –1 ribosomal frameshifting (–1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate –1 PRF. Through a reporter-based genome-wide CRISPR/Cas9 knockout screen, we identified several host factors that either suppressed or enhanced –1 PRF of SARS-CoV-2. One of these factors is eukaryotic translation initiation factor 2A (eIF2A), which specifically and directly enhanced –1 PRF in vitro and in cells. Consistent with the crucial role of efficient –1 PRF in transcriptase/replicase expression, loss of eIF2A reduced SARS-CoV-2 replication in cells. Transcriptome-wide analysis of eIF2A-interacting RNAs showed that eIF2A primarily interacted with 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results revealed an unexpected role for eIF2A in modulating the translation of specific RNAs independent of its previously described role during initiation.

2024-07-11 | GSE223690 | GEO

Genome-wide CRISPR screen for regulators of SARS-CoV-2 programmed ribosomal frameshifting

Project description:Genome-wide CRISPR-Cas9 knockout screens were performed in dual-fluorescent frameshifting reporter cell lines to identify human host factors for SARS-CoV-2 programmed ribosomal frameshfiting.

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Massively parallel reporter assay for programmed ribosomal frameshifting on designed sequence libraries

Project description:We designed an oligonucleotide libraries containing a potential frameshifting site, cloned the library variants into a dedicated reporter construct, transfected and integrated these constructs in the genome of K562 cells, and performed FACSseq (as well as targeted RNA sequencing) to determine if and to what extent frameshifting occurs.

2020-02-22 | GSE145684 | GEO

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Project description:Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

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Massively parallel reporter assay for programmed ribosomal frameshifting on designed sequence libraries

Project description:Massively parallel reporter assay for programmed ribosomal frameshifting on designed sequence libraries

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The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

Project description:Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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Genome-wide CRISPR screen for regulators of SARS-CoV-2 programmed ribosomal frameshifting

Project description:Genome-wide CRISPR screen for regulators of SARS-CoV-2 programmed ribosomal frameshifting

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Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

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Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human[ChIP-seq]

Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats,which may expand the coding capacity of mRNA and diversify the regulation in human.

2023-05-01 | GSE220676 | GEO

programmed ribosomal frameshift

Project description:programmed ribosomal frameshift Raw sequence reads

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