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Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes

ABSTRACT: Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes

PROVIDER: PRJNA894451 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22045942.fastq.gz	Fastqsanger.gz
	SRR22045943.fastq.gz	Fastqsanger.gz
	SRR22045944.fastq.gz	Fastqsanger.gz
	SRR22045945.fastq.gz	Fastqsanger.gz
	SRR22045946.fastq.gz	Fastqsanger.gz

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

Project description:Effective extraction of polyribosomes from primary astrocytes exposes strategies of gene expression regulation [Heavy_complex_RNAseq]

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

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

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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

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

Project description:We optimized a 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 Riboseq 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 in astrocytes 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 | GSE216580 | GEO

Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-02-09 | GSE216583 | GEO

Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes.

Project description:Regulation of mRNA translation in astrocytes gains a growing interest. However, until now, successful ribosome profiling of primary astrocytes has not been reported. Here, we optimized the standard 'polysome profiling' method and generated an effective protocol for polyribosome extraction, which enabled genome-wide assessment of mRNA translation dynamics along the process of astrocyte activation. Transcriptome (RNAseq) and translatome (Riboseq) data generated at 0, 24 and 48 h after cytokines treatment, revealed dynamic genome-wide changes in the expression level of ∼12 000 genes. The data clarify whether a change in protein synthesis rate results from a change in mRNA level or translation efficiency per se. It exhibit different expression strategies, based on changes in mRNA abundance and/or translation efficiency, which are specifically assigned to gene subsets depending on their function. Moreover, the study raises an important take-home message related to the possible presence of 'difficult to extract' polyribosome sub-groups, in all cell types, thus illuminating the impact of ribosomes extraction methodology on experiments addressing translation regulation.

| S-EPMC10123121 | biostudies-literature

A tomato fruit RNA extraction method that isolates mRNAs encoding secreted and endomembrane associated proteins

Project description:Background: During the analysis of ripening related gene expression in tomato fruit, we observed a bias towards certain classes of messenger RNA based upon the type of buffer used in the initial step of the extraction procedure. We postulated that there was a functional association of the separated transcripts. To test this hypothesis, we carried out extractions from the same tissue sample where only the buffer varied. Transcripts were hybridised to Affymetrix oligo arrays and the data was analysed according to the predicted cellular component of the encoded proteins. Results: The use of an extraction buffer that lacked high levels of caeotropic agents resulted in a reduction of mRNAs encoding proteins that were either secreted or were predicted to be routed through the endomembrane system and hence could have been bound to the endoplasmic reticulum in polyribosomes. Extraction of the cell debris immediately following the initial buffer based extraction and subsequent micro array analysis revealed that the expected transcripts could be recovered. This demonstrated that the buffer was separating the mRNAs based on the cellular component of the encoded proteins. Analysis of selected transcripts by northern hybridisation again supported this theory. Conclusions: Some traditional buffers used for fruit RNA extraction selectively deplete for transcripts encoding proteins that are membrane-associated or secreted. This can be explained if polyribosomes that are bound to the endoplasmic reticulum (ER) are not effectively disrupted when the extraction buffer lacks either detergent or organic solvents. These findings have important implications with respect to experimental bias, as well as opportunities for message enrichment and protein characterisation.

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Effect of miR-122 overexpression of mRNA distribution on the polyribosomes

Project description:The aim of this study was to identify at genome-wide level, the effect of miR-122 on the distribution of its responsive genes on the polyribosomes. For this purpose human hepatoma cells Huh-7 were transfected for 48 hrs with LNA-based miR-122 inhibitors (122-PM) or miR-122 mimics (122-MIM). Following the treatment with cycloheximide, cells were lysed and layered on a 10-50% sucrose gradient. Following ultracetrifigation the polyribosomes were separated in 12 different fractions

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A tomato fruit RNA extraction method that isolates mRNAs encoding secreted and endomembrane associated proteins

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Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

Project description:Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein “bait”. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs wes analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

2018-10-20 | GSE121524 | GEO

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