A global overview of translation efficiency and ribosome occupancy at nucleotide resolution in HEK293 cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA556704 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9839460.fastq.gz | Fastqsanger.gz | |||
| SRR9839461.fastq.gz | Fastqsanger.gz | |||
| SRR9839462.fastq.gz | Fastqsanger.gz | |||
| SRR9839463.fastq.gz | Fastqsanger.gz | |||
| SRR9839464.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We have developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing (Baltz and Munschauer et al. 2012). Our current work focuses on streamlining and extending protein occupancy profiling on poly(A)-RNA. Our objectives are to identify previously unknown protein-bound transcripts and, more importantly, to assess global and local differences in protein occupancy across different biological conditions. To this end, we have implemented poppi, the first pipeline for differential analysis of protein occupancy profiles. We have applied our analysis pipeline to pinpoint changes in occupancy profiles of MCF7 cells against already published HEK293 cells [GSE38157].
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Project description:The alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance(AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we benchmarked a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance(ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ~1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.
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