Long-RNA sequencing and ribosome profiling of inflamed beta-cells reveal an extensive translatome landscape
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE167223 | GEO | 2021/02/23
REPOSITORIES: GEO
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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:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.
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Project description:Unorthodox rules of extracting genetic information enable proteome expansion without increasing the genome size. The use of alternative translation initiation sites achieves this goal by allowing production of more than one protein from a single gene. Although several such examples have been serendipitously found in bacteria, genome-wide experimental mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin treatment followed by Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the annotated Escherichia coli genes but, strikingly, it also revealed putative alternative internal start sites in a number of genes. Experimental evidence demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose translation is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. In addition to proteome expansion, the internal start sites may play regulatory role in gene expression.
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Project description:Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor C/EBPα produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans-acting factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBPα isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.
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Project description:Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor C/EBPα produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans-acting factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBPα isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.
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Project description:Cellular stress is often accompanied by non-canonical initiation of translation at alternate start codons in mammalian cells. Here we systematically investigate the extent and impact of alternate translation initiation in the context of influenza virus infection. We use ribosome profiling with the initiation inhibitor lactidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus.
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Project description:Regulated translation initiation has the potential to reshape the proteome, but conditions under which start codon selection is altered remain poorly defined. Here, using global translation initiation site profiling, we reveal widespread changes in start codon selection during the mammalian cell cycle. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in changes in the relative translation of thousands of annotated proteins, alternative translational isoforms, and uORFs. This increased mitotic translational stringency results from the cytoplasmic release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Increased eIF1-40S ribosome interactions repress suboptimal initiation sites to rewire the mitotic proteome. Selectively depleting the nuclear pool of eIF1 eliminates changes to translational stringency during mitosis, resulting in substantially increased cell death following an extended mitotic delay induced by anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency to alter their physiology with critical roles during the mammalian cell cycle.
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Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
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Project description:Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of GGGGCC; these sites are detected irrespective of the presence or absence of the repeat.
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Project description:Long-RNA sequencing and ribosome profiling of inflamed beta-cells reveal an extensive translatome landscape
| PRJNA703954 | ENA