Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA193306 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR786387_1.fastq.gz | Fastqsanger.gz | |||
| SRR786387_2.fastq.gz | Fastqsanger.gz | |||
| SRR786388_1.fastq.gz | Fastqsanger.gz | |||
| SRR786388_2.fastq.gz | Fastqsanger.gz | |||
| SRR786391_1.fastq.gz | Fastqsanger.gz |
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Project description:Transcription and translation correlate poorly, as mRNA undergoes multiple regulatory steps such as alternative splicing and microRNA regulation that determine its translationability into protein. Measures of transcriptional mRNA levels may therefore misrepresent cellular activation. To test this hypothesis employing human physiological mRNA levels, we analyzed cytoplasmic and polyribosome-bound mRNA expression (Frac-seq) combined with microRNA profiling by small RNA-seq in bronchoepithelial cells from healthy and severe asthma (SA) donors, SA being a chronic inflammatory airways disease, poorly understood at the molecular level. We found 275 genes bound to polyribosomes differentially between healthy and severe asthma, of which only 11.64% overlapped with differentially expressed cytoplasmic mRNAs (226 genes). We found 335 alternatively spliced mRNA isoforms differentially bound to polyribosomes of which only ~8% were revealed by cytoplasmic mRNA analysis. Approximately two thirds of differentially expressed isoforms could not be found at the gene level in both cytoplasmic and polyribosome bound fractions, demonstrating the disruption of splicing in asthma. Only the analysis of differentially expressed isoforms bound to polyribosomes revealed disease-related pathways overlooked in total mRNA. We detected a network of 21 microRNAs differentially expressed, with 8 microRNAs accounting for more than 80% targeting observed in both cytoplasmic and polyribosome bound mRNA isoforms. Importantly, microRNAs target distinct cytoplasmic and polyribosome bound mRNAs. Hence this work, integrating deep-sequencing, subcellular fractionation and microRNA profiling, demonstrates the disruption of post-transcriptional regulatory processes as the main disease causing molecular mechanism in asthma, something that cannot be dissected employing more classic transcriptomics approaches alone.
2018-04-11 | GSE85215 | GEO
Project description:Transcription and translation correlate poorly, as mRNA undergoes multiple regulatory steps such as alternative splicing and microRNA regulation that determine its translationability into protein. Measures of transcriptional mRNA levels may therefore misrepresent cellular activation. To test this hypothesis employing human physiological mRNA levels, we analyzed cytoplasmic and polyribosome-bound mRNA expression (Frac-seq) combined with microRNA profiling by small RNA-seq in bronchoepithelial cells from healthy and severe asthma (SA) donors, SA being a chronic inflammatory airways disease, poorly understood at the molecular level. We found 275 genes bound to polyribosomes differentially between healthy and severe asthma, of which only 11.64% overlapped with differentially expressed cytoplasmic mRNAs (226 genes). We found 335 alternatively spliced mRNA isoforms differentially bound to polyribosomes of which only ~8% were revealed by cytoplasmic mRNA analysis. Approximately two thirds of differentially expressed isoforms could not be found at the gene level in both cytoplasmic and polyribosome bound fractions, demonstrating the disruption of splicing in asthma. Only the analysis of differentially expressed isoforms bound to polyribosomes revealed disease-related pathways overlooked in total mRNA. We detected a network of 21 microRNAs differentially expressed, with 8 microRNAs accounting for more than 80% targeting observed in both cytoplasmic and polyribosome bound mRNA isoforms. Importantly, microRNAs target distinct cytoplasmic and polyribosome bound mRNAs. Hence this work, integrating deep-sequencing, subcellular fractionation and microRNA profiling, demonstrates the disruption of post-transcriptional regulatory processes as the main disease causing molecular mechanism in asthma, something that cannot be dissected employing more classic transcriptomics approaches alone.
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Project description:Alternative splicing (AS) influences the expression of human genes in diverse ways. We previously used subcellular fraction-sequencing (Frac-Seq) to reveal an unexpected connection between alternative splicing and isoform-specific mRNA translation. Here we apply comparative transcriptomics to explore alternative splicing coupled translational control (AS-TC) across 13 million years of primate evolution. We used Frac-seq to identify polyribosome associated mRNA isoforms from human, chimpanzee and orangutan induced pluripotent stem cell lines. We discovered orthologous AS-TC events with either conserved or species-specific translation patterns. Exons sequences associated with similar sedimentation profiles between species show strong sequence conservation compared to orthologous exons with divergent sedimentation profiles, suggesting exonic cis-regulatory elements influence to translational control. To test this hypothesis we created luciferase reporters from orthologous exons with divergent sedimentation profiles differing by a single nucleotide. Remarkably, single nucleotide substitutions were sufficient to drive species-specific expression of luciferase reporters. Together these data establish that cis-acting elements regulate AS-TC across primate species.
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Project description:The goal of this study was to test the hypothesis that minor alternative mRNA isoforms can be preferentially translated. Included are Cytoplasm and Polyribosome samples in duplicate from both Control and Rapamycin treated cells.
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Project description:Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESC and NPC respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long-read sequencing-based method to study translational control.
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Project description:The goal of this study was to test the hypothesis that minor alternative mRNA isoforms can be preferentially translated.
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Project description:The Fragile X Mental Retardation Protein, FMRP, is thought to regulate the translation of a specific set of neuronal mRNAs on polyribosomes. Therefore, we prepared polyribosomes on sucrose gradients and purified mRNA specifically from these fractions, as well as the total mRNA levels, to determine whether a set of mRNAs might be changed in its % association with polyribosomes in the absence of FMRP in the KO mouse model. No significant differences were found, other than the Fmr1 transcript itself, in total mRNA levels or % polyribosome association that withstood multiple test correction, in P7 Fmr1 KO mouse cerebral cortex compared with WT littermates..
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