Optimization of Ribosome Profiling Using Brain Tissue from Fragile X Mice
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA477831 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7421911.fastq.gz | Fastqsanger.gz | |||
| SRR7421912.fastq.gz | Fastqsanger.gz | |||
| SRR7421913.fastq.gz | Fastqsanger.gz | |||
| SRR7421914.fastq.gz | Fastqsanger.gz | |||
| SRR7421915.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 56 |
Similar Datasets
Project description:Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases such as Fragile X Syndrome. A very robust technique is required to capture subtle but biologically significant differences in neurological disorders. Ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease digestion by ribosomes, is a powerful tool to study translational control. However, it has been mainly applied to rapidly dividing cells where translation is robust and where large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and where gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for ribonuclease digestion. We performed ribonuclease titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling especially for low-input brain tissue.
2019-03-15 | GSE116233 | GEO
Project description:Fragile X syndrome (FXS) is caused by inactivation of FMR1 gene and loss of its encoded product the RNA binding protein FMRP, which generally represses translation of its target transcripts in the brain. In mouse models of FXS (i.e., Fmr1 knockout animals; Fmr1 KO), deletion of Cpeb1, which encodes a translational activator, mitigates nearly all pathophysiologies associated with the disorder. Here we reveal unexpected wide-spread dys-regulation of RNA abundance in Fmr1 KO brain cortex and its rescue to normal levels in Fmr1/Cpeb1 double KO mice. Alteration and restoration of RNA levels are the dominant molecular events that drive the observed dys-regulation and rescue of translation as measured by whole transcriptome ribosome occupany in the brain. The RNAs down-regulated and rescued in these animal models are highly enriched for FMRP binding targets and have an optimal codon bias that would predict their stability in wild type and possible instability in FMRP knock-out brain. These results leads to a further study to profile RNA metabolism rates in Fragile X neurons.
2020-06-01 | GSE140565 | GEO
Project description:Ribosome profiling and RNA-seq of Fragile X mouse brain cortex
| PRJNA590189 | ENA
Project description:Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for RNase digestion. We performed RNase titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling for low-input brain tissue.
| S-EPMC6411937 | biostudies-literature
Project description:Fragile X syndrome (FXS) is caused by inactivation of FMR1 gene and loss of its encoded product the RNA binding protein FMRP, which generally represses translation of its target transcripts in the brain. In mouse models of FXS (i.e., Fmr1 knockout animals; Fmr1 KO), deletion of Cpeb1, which encodes a translational activator, mitigates nearly all pathophysiologies associated with the disorder. We have observed that the wide-spread dys-regulation of RNA abundance in Fmr1 KO brain cortex and its rescue to normal levels in Fmr1/Cpeb1 double KO mice were the driver of the observed dys-regulation and rescue of translation as measured by whole transcriptome ribosome occupany in the brain. We hypothesize that in Fragile X brain there is wide spread dys-regulation at RNA stability level. Here we test this hypothesis by profiling RNA synthesis, processing and degradation rates in Fragile X and wild type neurons, by taking advantage of short 5-EU labeling and computational modeling. We show that, while RNA synthesis and processing rates were barely changed, there is wide-spread evelated RNA degradation rates in the Fragile X neurons, particularly for genes using optimal codons.
2020-06-01 | GSE140642 | GEO
Project description:Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not PABPC1, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of the putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes and can stimulate NGD in cells, albeit on a small number of transcripts, revealing an unappreciated role of FMRP that would be misregulated in FXS when FMRP is lost.
2024-08-28 | GSE254586 | GEO
Project description:Optimization hydrophobic tagging reaction time (30 min to 2h) and evaluation of the HUNTER protocol performance with different amounts (25 ug to 400 ug) of purified rat brain proteome as starting material.
2019-09-03 | PXD012844 | Pride
Project description:Silencing of FMR1 and loss of its gene product FMRP results in Fragile X Syndrome. FMRP binds brain mRNAs and inhibits polypeptide elongation. Using ribosome profiling of the hippocampus, we find that ribosome footprint levels in Fmr1-deficient tissue mostly reflect changes in RNA abundance. Profiling over a time course of ribosome runoff in wildtype tissue reveals a wide range of ribosome translocation rates; on many mRNAs, the ribosomes are stalled. Sucrose gradient ultracentrifugation of hippocampal slices after ribosome runoff reveals that FMRP co-sediments with stalled ribosomes; and its loss results in decline of ribosome stalling on specific mRNAs. One such mRNA encodes SETD2, a lysine methyltransferase that catalyzes H3K36me3. ChIP-Seq demonstrates that loss of FMRP alters the deployment of this epigenetic mark on chromatin. H3K36me3 is associated with alternative pre-RNA processing, which we find occurs in an FMRP-dependent manner on transcripts linked to neural function and autism spectrum disorders.
2020-03-31 | GSE143331 | GEO
Project description:Silencing of FMR1 and loss of its gene product FMRP results in Fragile X Syndrome. FMRP binds brain mRNAs and inhibits polypeptide elongation. Using ribosome profiling of the hippocampus, we find that ribosome footprint levels in Fmr1-deficient tissue mostly reflect changes in RNA abundance. Profiling over a time course of ribosome runoff in wildtype tissue reveals a wide range of ribosome translocation rates; on many mRNAs, the ribosomes are stalled. Sucrose gradient ultracentrifugation of hippocampal slices after ribosome runoff reveals that FMRP co-sediments with stalled ribosomes; and its loss results in decline of ribosome stalling on specific mRNAs. One such mRNA encodes SETD2, a lysine methyltransferase that catalyzes H3K36me3. ChIP-Seq demonstrates that loss of FMRP alters the deployment of this epigenetic mark on chromatin. H3K36me3 is associated with alternative pre-RNA processing, which we find occurs in an FMRP-dependent manner on transcripts linked to neural function and autism spectrum disorders.
2020-03-31 | GSE143330 | GEO