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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE216580 | GEO | 2023/02/09

REPOSITORIES: GEO

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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.

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Project description:While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

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Analysis of mitochondrial gene expression with Ribosome Profiling

Project description:Ribosome profiling (RiboSeq) maps positions of translating ribosomes on the transcriptome. Here we optimized ribosome profiling for footprinting mitochondrial ribosomes, and profiled three human cell-lines - HEK293, a PDE12-/- knockout, and a deltaFLP 143B cybrid.

2017-07-25 | E-MTAB-5519 | biostudies-arrayexpress

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

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Project description:An optimized protocol for retina single-cell RNA sequencing

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High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

Project description:We developed a high-throughput sequencing protocol that efficiently captures small RNAs from low input samples while minimizing inherent biases associated with library production. The protocol was builds on previously published method that adds a barcode to samples at the earliest step in preparation such that all downstream manipulations can be performed on a pool of many samples thereby greatly reducing reagent usage and workload. We optimized adapter concentrations and showed modified adapters greatly increases the efficiency of small RNA capture. This work also demonstrates that unique molecular identifiers incorporated into the adapters can be used to bioinformatically remove PCR duplication.

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2020-09-23 | GSE157852 | GEO

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