Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single cell quantification of ribosome occupancy in early mouse development

ABSTRACT: Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts with allelic-biased expression. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE185732 | GEO | 2023/02/06

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

tsRibo-Seq: An Approach to Zoom In into Translation of Single Transcripts

Project description:Ribo-Seq (ribosome profiling) enables global assessment of translation efficiency, yields and fidelity. However, low-abundance transcripts receive little coverage and fall outside of the Ribo-Seq detection limit. We developed a new twist of the Ribo-Seq approach, termed transcript specific Ribo-Seq (tsRibo-Seq) to specifically detect lowly abundant messenger RNAs (mRNAs). This approach yields unprecedented depth in the coverage of individual low-abundance transcripts allowing identification of slow and fast translating regions with nucleotide resolution.

2020-10-03 | GSE104587 | GEO

Evolution of Translation Regulation in budding Yeast

Project description:Comparison of translation efficiency in S. cerevisiae, S. paradoxus, and their F1 hybrid. SRA submission number SRP028552; BioProject number PRJNA213844; Ribosome profiling was used to compare mRNA abundance, ribosome occupancy, and translation efficiency in two yeast species and their F1 hybrid.

2013-12-04 | E-GEOD-52119 | biostudies-arrayexpress

Synonymous codon usage regulates translation initiation

Project description:Ribo-seq with firefly luciferase optimality reporters to determine whether incomplete translation and/or inhibited translation initiation is responsible for the reduced ribosome occupancy on nonoptimal transcripts.

2022-05-16 | GSE202900 | GEO

Ribosome profiling of Japanese encephalitis virus infected Neuro2a cells

Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of mouse neuroblastoma (Neuro2a) cells infected with Japanese Encephalitis Virus (JEV) P20778 Vellore strain. At 18 h post infection (p.i.), infected cells were treated with either cycloheximide (translation elongation inhibitor) or in combination with harringtonine (translation initiation inhibitor). Ribo-Seq libraries were prepared using a broad range of fragment lengths (25 to 70 nucleotides) and deep sequenced. This allowed for estimation of ribosomal frameshifting efficiency and discovery of a novel upstream open reading frame (uORF) in JEV along with perturbations in ribosome-associated tRNA levels upon JEV infection.

2021-07-05 | E-MTAB-9352 | biostudies-arrayexpress

Assembly and functionality of the ribosome with tethered subunits

Project description:Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171), RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.

2019-02-25 | GSE119454 | GEO

Ribosome Profiling and Mass Spectrometry Reveal Widespread Mitochondrial Translation Defects in a Striatal Cell Model of Huntington Disease

Project description:Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear encoded mitochondrial transcripts involved in oxidative phosphorylation (OXPHOS) (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded OXPHOS mRNAs (mtNd-1, mtNd-2, mtNd-4, mtNd-4l, mtNd-5, mtNd-6, mt-Co1, mtCyt b, and mt-ATP8). We also applied tandem mass tag based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein turnover of mitochondria related genes in HD.

| MSV000092843 | MassIVE

Effective extraction of polyribosomes from primary astrocytes exposes strategies of gene expression regulation [RNAseq_Riboseq]

Project description:We optimized a standard polysome profiling protocol considering the unique morphology and ribosomal distribution of astrocytes. This ‘astrocyte-optimized’ protocol was then used to study primary astrocytes treated with proinflammatory cytokines, enabeling Ribo-seq analysis for measurment of the transcriptome, translatome, and translation efficiency with a coverage of over 12,000 genes. We found several regulation strategies of gene expression executed as part of an extensive inflamatory response. Enhanced translation efficiency was one of these strategies, used specifically for genes related to oxidative phosphorylation, and ribosomal proteins.

2023-02-09 | GSE216582 | GEO

hnRNP C is a key regulator of protein synthesis in mitosis

Project description:Purpose: The goal of this study is to characterize the role of hnRNP C in translation regulation of a specific set of mRNAs during mitosis. Methods: mitotic HeLa S3 cells expressing a Dox-inducible shRNA against hnRNP C were used and profiles of total mRNAs and ribosome footprints (RFs) of either untreated (wild type) cells or hnRNP C-knockdown cells were generated by deep sequencing, using Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to a reference set of human curated protein‐coding transcripts (plus the five human rRNA transcripts) using bowtie2. Expression estimates were further normalized using quantile normalization. qRT–PCR validation was performed using SYBR Green assays. Results: RNA‐seq and Ribo/RF‐seq datasets were combined according to gene ID. Only genes with expression level of at least 1.0 RPKM in both datasets were included in subsequent analyses. The combined dataset includes 10,996 genes. Transcripts encoding components of the translation machinery (Gene Ontology Biological Process [GOBP] “Translation” annotation) show reduced ribosome occupancy during mitosis under hnRNP C knockdown (p-value = 1.07 x 10-13) although their total mRNA levels are increased (p-value = 1.78 x 10-5). Translation efficiency (TE) calculations of ribosome occupancy per mRNA level showed a statistically-significant reduction for mRNAs encoding ribosomal proteins (Gene Ontology Cellular Compartment [GOCC] “Ribosome” annotation; p-value = 9.92 x 10-12). In particular, the TE of 5’ TOP-containing mRNAs was found to be significantly reduced under hnRNP C knockdown (p-value = 5.16 x 10-4). Conclusions: Our study represents the first detailed analysis of the translation effeciency of mRNAs in wild type and hnRNP C knockdown HeLa S3 cells during mitosis using RNA-Seq and Ribo-Seq. Our results show that the translation of a specific subset of mRNAs (5' TOP mRNAs) is regulated by hnRNP C in mitosis.

2017-06-23 | GSE83493 | GEO

Genome-Wide Translation Profiling by Ribosome-Bound tRNA Capture

Project description:In the ribosome complex, tRNA is a critical element of mRNA translation. We reported a new technology for profiling ribosome-embedded tRNAs and their modifications. With the method, we generated a comprehensive survey of the quanity and quality of intra-ribosomal tRNAs (Ribo-tRNA-seq). Ribo-tRNA-seq can provide new insights on translation control mechanism in diverse biological contexts.

2018-03-19 | E-MTAB-6576 | biostudies-arrayexpress

Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq

2014-08-01 | E-GEOD-59573 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data