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Redefining the translational status of 80S monosomes

ABSTRACT: Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE76117 | GEO | 2016/02/11

SECONDARY ACCESSION(S): PRJNA306259

REPOSITORIES: GEO

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Redefining the translational status of 80S monosomes

Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.Â We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.

2016-02-11 | E-GEOD-76117 | biostudies-arrayexpress

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5'UTR and Responsive to mTORC1 Signaling (RNA-Seq)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5M-bM-^@M-^Y untranslated region (5M-bM-^@M-^Y UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Monitor the translational status of transcriptome in mammalian cells under different conditions

2011-11-12 | E-GEOD-25626 | biostudies-arrayexpress

Monosomes actively translate synaptic mRNAs in neuronal processes

Project description:In order to deal with their huge volume and complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. A relative paucity of polyribosomes (considered the active sites of translation) detected in electron micrographs of neuronal processes (axons and dendrites), however, has suggested a rather limited capacity for local protein synthesis. Polysome profiling together with ribosome footprinting of microdissected synaptic regions revealed that a surprisingly high number of dendritic and/or axonal transcripts were predominantly associated with monosomes (single ribosomes). Contrary to prevailing views, the neuronal monosomes were in the process of active protein synthesis (e.g. they exhibited elongation). Most mRNAs showed a similar translational status in the somata and neuropil, but some transcripts exhibited differential ribosome occupancy in the compartments. Strikingly, monosome-preferring transcripts often encoded high-abundance synaptic proteins. These data suggest a significant contribution of monosome translation to the maintenance of the local neuronal proteome. This mode of translation can presumably solve some of restricted space issues (given the large size of polysomes) and also increase the diversity of proteins made from a limited number of ribosomes available in dendrites and axons.

2020-01-27 | PXD016552 | Pride

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling (Microarray)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway.

2011-11-12 | GSE25331 | GEO

Saccharomyces cerevisiae

Project description:Redefining the translational status of 80S monosomes

| PRJNA306259 | ENA

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5'UTR and Responsive to mTORC1 Signaling (RNA-Seq)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway.

2011-11-12 | GSE25626 | GEO

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling (Microarray)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Untreated TSC2 WT MEFs, TSC2 KO MEFs and TSC2 WT MEFs, TSC2 KO MEFs treated with 20nM rapamycin for 30 minutes or 3hours were harvested for ribosme profiling. The fraction samples were pooled into three groups based on velocity sedimentation: single ribosome fraction (Small group), fractions with 2 ~ 4 ribosomes (Medium group), and the one with ≥5 ribosomes (Large group). RNA were extracted from the whole cell lysis and each fraction group.

2011-11-12 | E-GEOD-25331 | biostudies-arrayexpress

Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1 at the A site

Project description:In response to environmental changes, cells flexibly and rapidly alter gene expression, through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is upregulated through upstream open reading frames (uORFs) that comprise only AUG and stop codons, in response to a shortage of boric acid in the environment. However, the molecular details of how the minimum uORF controls the translation of the downstream main ORF, in a boric acid concentration-dependent manner, have remained unclear. Here, combining ribosome profiling, TCP-Seq, structural analysis with cryo-electron microscopy, and biochemical assays, we showed that the 80S ribosome (80S) assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 (eRF1) on the 80S on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.

2023-09-19 | GSE223755 | GEO

eIF3 associates with 80S ribosomes to promote translation elongation, mitochondrial homeostasis, and muscle health

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.

2020-06-15 | GSE149697 | GEO

Boric acidon intercepts 80S ribosome migration from AUG-stop by stable eRF1 immobilization

Project description:Upon the environmental changes, cells flexibly and rapidly alter the gene expression by the translation control. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is upregulated through the upstream open reading frame (uORF), which comprises only AUG and stop codons, in response to the shortage of boric acid. However, the molecular details of how the minimum uORF controls the translation of downstream main ORF depending on the boric acid concentration remained unclear. Here we showed that the 80S ribosome (80S) assembled at AUG-stop migrates into the subsequent RNA segment for downstream translation initiation and that the presence of boric acid impedes the process by stable confinement of eukaryotic release factor 1 (eRF1) on 80S on AUG-stop. Ribosome profiling along the in vitro-translated reporter mRNA revealed that the number of the ribosomes on AUG-stop and that along the downstream are anti-correlated in response to boric acid, reflecting the 80S scanning leaving AUG-stop for translation initiation from downstream ORF. Moreover, cryo-electron microscopy (cryo-EM) analysis revealed that, in the presence of boric acid, the initiator methionyl-tRNA (Met-tRNAi) in the P site stably contacts eRF1 in the A site on AUG-stop. In contrast, the absence of boric acid led to weaker eRF1 density, suggesting that eRF1 cannot be stably positioned without boric acid. Consistent with the structural features, the hydrolysis of Met-tRNAi on AUG-stop was accelerated by boric acid, which is likely to be the checkpoint for the subsequent 80S scanning of the downstream. Our results provide a molecular insight into the translation regulation by a minimum and environment-responsive uORF.

2023-09-20 | GSE189222 | GEO

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