Low input ribosome profiling reveals stage-specific translational regulation during early C. elegans embryogenesis
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ABSTRACT:
PROVIDER: PRJNA1183817 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31283500.fastq.gz | Fastqsanger.gz | |||
| SRR31283501.fastq.gz | Fastqsanger.gz | |||
| SRR31283502.fastq.gz | Fastqsanger.gz | |||
| SRR31283503.fastq.gz | Fastqsanger.gz | |||
| SRR31283504.fastq.gz | Fastqsanger.gz |
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Project description:Translation of maternal mRNAs guides early embryonic development. C. elegans embryos exhibit an invariant cell lineage from the first division, offering a unique opportunity to trace the impact of post-transcriptional gene regulation on subsequent embryonic development. Although prior work has explored the translation of a select few transcripts, we lack a transcriptome-wide view of translation control during early embryogenesis. We developed a method for low-input ribosome profiling and combined it with low-input RNA sequencing to measure new protein translation during the first four cell cycles of C. elegans embryogenesis. Strikingly, our results show a poor correlation between RNA abundance and ribosome occupancy in this system compared to other model systems, emphasizing the predominant role of variation in translation efficiency in this system. We found numerous translationally regulated transcripts that had not been previously identified. Housekeeping genes involved in ribosome function and biogenesis were stably translated throughout the studied stages, while genes involved in mRNA processing and mRNA binding are dynamically regulated, consistent with roles in cell fate specification. A gain-of-function mutation in a known translational repressor protein, OMA-1, stalls the developmental program by preventing the translational remodeling that should occur at the 2-cell stage. These insights advance our understanding of the post-transcriptional regulatory mechanisms that shape the embryonic proteome and direct cell differentiation, and provide a valuable resource studies of C. elegans development.
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Project description:In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we employed Ribo-ITP, a low input ribosome profiling technique on difficult to collect samples like microdissected hippocampal tissues and single pre-implantation embryos. We detected thousands of translons across these samples, most of which initiated at non-canonical start sites, were less than 100 amino long with significantly altered amino acid composition. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed pervasive expression of most of the detected translons. To test the translational capacity of the detected translons, we engineered a translon dependent GFP reporter system, and detected robust expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from ultra-low input samples which can be applied to cell and tissue types inaccessible to conventional methods.
2025-08-05 | GSE300832 | GEO
Project description:In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we employed Ribo-ITP, a low input ribosome profiling technique on difficult to collect samples like microdissected hippocampal tissues and single pre-implantation embryos. We detected thousands of translons across these samples, most of which initiated at non-canonical start sites, were less than 100 amino long with significantly altered amino acid composition. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed pervasive expression of most of the detected translons. To test the translational capacity of the detected translons, we engineered a translon dependent GFP reporter system, and detected robust expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from ultra-low input samples which can be applied to cell and tissue types inaccessible to conventional methods.
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Project description:Ribo-ITP coupled ribosome profiling identifies non-canonical translational events in low input samples
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Project description:MicroRNAs regulate gene expression through deadenylation, repression and mRNA decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild type and dicer mutants lacking mature miR-430. Our results indicate that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation using an internal poly(A) tail did not block target repression. Finally, we observe that ribosome density along the length of the target mRNA remains constant, suggesting that translational repression occurs by reducing the initiation rate rather than reducing elongation or causing ribosomal drop-off. In summary, our results show that miR-430 regulates translation initiation before inducing mRNA decay. Time course parallel ribosome profiling and input mRNA quantification in wildtype and MZdicer mutant embryos
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Project description:Ribo-ITP coupled ribosome profiling identifies non-canonical translational events in low input samples [Ribo-seq]
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Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655
2016-01-13 | E-GEOD-72899 | biostudies-arrayexpress
Project description:Translational control is a widespread mode of gene regulation in organisms ranging from bacteria to mammals. Computational models posit that translational control of protein expression during elongation is exerted through a traffic jam of multiple ribosomes at ribosome pause sites on mRNAs. Yet neither the in vivo frequency of ribosome traffic jams nor the contribution of such traffic jams to protein expression has been measured in any organism. Here we show that upon starvation for single amino acids in the bacterium Escherichia coli, ribosome traffic jams are pervasive across the transcriptome, but they occur at only a subset of codons cognate to the limiting amino acid, and their severity is determined by the translation efficiency of mRNAs. Surprisingly, a computational model based on the observed traffic jams at ribosome pause sites is quantitatively inconsistent with measured protein synthesis rates. By comparison, a model incorporating abortion of protein synthesis at ribosome pause sites in addition to ribosome traffic jams predicts protein synthesis rate with higher accuracy. Consistent with the latter model, a significant fraction of the nascent polypeptides at ribosome pause sites is degraded through the activity of the transfer-messenger RNA during amino acid starvation in E. coli. Our work provides a minimal, experimentally-constrained model for predicting protein expression from ribosome dynamics, and it suggests the existence of a trade-off between the cellular translational capacity and the processivity of protein synthesis in vivo. 6 samples for ribosome profiling and 5 samples for total mRNA profiling
2014-05-15 | E-GEOD-51052 | biostudies-arrayexpress
Project description:Purpose: The aim is to analyze the translational kinetics of the basal and LPS-stimulated conditions of RAW264 macrophages focusing on ribosome density Method: RAW264 macrophages were cultured at 3.0 x 10^5 cells/ml in media (DMEM, 2mM Glutamine, 10% FBS, 100 units penicillin and 100µg streptomycin/mL) 24 hours prior to harvest. It was confirmed that the confluency of cells never surpassed 80 ~ 90%. Cell were harvested in their basal state or LPS stimulated condition (100 ng/ml for 30 min) and ribosome profiling (Ribo-Seq) and high-throughput mRNA sequencing (mRNA-Seq) were conducted. Ribosome protected fragments (RPF) were sequenced and the density was normalized by mRNA abundance. Result and conclusion: We discovered where in the ribosome translational arrest ocurs and how this happens. Translational stall was striking at A-site and P-site of ribosomal complex. The other arrest site was observed 3 to 5 residues away from the peptidyl transfer center of the exit tunnel, in which ribosomal density and hydrophobicity showed a significant negative correlation. mRNA and RPF of RAW264 macrophages were deep-sequenced with two independent biological replicates by Ion PGM sequencer
2015-11-01 | E-GEOD-60930 | biostudies-arrayexpress
Project description:Translational regulation plays a critical role during oocyte and preimplantation embryo development. Here, we utilized high resolution ribosome fractionation and low-input ribosome profiling of mouse oocytes and preimplantation embryos to explore the translational landscapes of early embryo development. The inconsistency was observed when comparing polysome-bound, monosome-bound, and free RNA profiles, indicating the role of ribosomes in manipulating RNA fate, especially for oocyte-to-embryo transition (OET) and zygotic genome activation (ZGA). Moreover, eif1ad3 with its homologs, including eif1ad4, eif1ad6, eif1ad7, etc. are highly translated during ZGA and regulating the translational activity of other genes. Knocking down these genes lead to defective ZGA and preimplantation development. These data reveal that the transcriptome and translatome manipulate RNA fate collectively to regulate mouse preimplantation development.
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