Bi-directional ribosome scanning controls the stringency of start codon selection
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA734658 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14714407.fastq.gz | Fastqsanger.gz | |||
| SRR14714408.fastq.gz | Fastqsanger.gz | |||
| SRR14714409.fastq.gz | Fastqsanger.gz | |||
| SRR14714410.fastq.gz | Fastqsanger.gz | |||
| SRR14714411.fastq.gz | Fastqsanger.gz |
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Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
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Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5'→3' migration of the pre-initiation complex (PIC) along the 5' UTR. In probing translation initiation from ultra-short 5' UTR, we report that an AUG triplet near the 5' end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5' end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5' end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
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Project description:Regulated start-codon selection has the potential to reshape the proteome through the differential production of upstream open reading frames, canonical proteins, and alternative translational isoforms. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation-initiation-site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start-codon selection during mitosis results from increased association between the 40S ribosome and the key regulator of start-codon selection, eIF1. We find that increased eIF1–40S ribosome interaction during mitosis is mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the change to translational stringency during mitosis, resulting in altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapies. Thus, cells globally control stringency of translation initiation, which has critical roles during the mammalian cell cycle in preserving mitotic cell physiology.
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Project description:Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of human genes, we identified 5 Hox gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. We analyzed published CAGE-seq data and generated CAGE-seq data from mRNAs from mouse somites. These data demonstrate that the 5’ leaders of Hox mRNAs of interest contain conserved uORFs, are much shorter than reported, and lack previously proposed IRES elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.
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Project description:Regulated start codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start codon selection during mitosis results from increased associations between the 40S ribosome and the key regulator of start codon selection, eIF1. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in the altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.
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Project description:Regulated start codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start codon selection during mitosis results from increased associations between the 40S ribosome and the key regulator of start codon selection, eIF1. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in the altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.
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