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Identifying the translatome of mouse NEBD – stage oocytes via SSP-profiling a novel polysome fractionation method

ABSTRACT: Identifying the translatome of mouse NEBD – stage oocytes via SSP-profiling a novel polysome fractionation method

PROVIDER: PRJNA497118 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8066570_1.fastq.gz	Fastqsanger.gz
	SRR8066570_2.fastq.gz	Fastqsanger.gz
	SRR8066571_1.fastq.gz	Fastqsanger.gz
	SRR8066571_2.fastq.gz	Fastqsanger.gz
	SRR8066572_1.fastq.gz	Fastqsanger.gz

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Identifying the translatome of mouse NEBD – stage oocytes via SSP-profiling; a novel polysome fractionation method

Project description:Meiotic maturation of oocyte relies on pre-synthesized maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol utilising small SW55Ti ultracentrifugation tubes with a volume of 5 ml. This newly optimized method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD); a developmental stage that has thus far remained unexplored. Our analyses have identified 1778 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteoasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we have also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

2020-02-12 | GSE121358 | GEO

Identifying the Translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling)

Project description:RNA-binding proteins (RBPs) have essential functions during oocyte development.In order to explore the regulatory role of RNA-binding protein LSM14B in oocyte, we Identified the translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling).

2023-09-08 | GSE206270 | GEO

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method.

Project description:Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

| S-EPMC7072993 | biostudies-literature

Polysome profiling of Arabidopsis translatome upon hypertonic stress

Project description:We examined the possible effects of hypertonic stress on Arabidopsis translatome using polysome profiling. We found that the translatome is partly and rapidly reprogrammed in response to hypertonic stress, and such translatome reprogramming is DCP5-dependent.

2022-01-20 | GSE193804 | GEO

Membrane-polysome associated fractionation of HES2 embryonic stem cells

Project description:The membrane fraction and the cytosolic fraction of HES2 cells were collected and subjected to microarray. The experiment was performed in triplicate Keywords: membrane-polysome fractionation of RNA Three consecutive passages of HES2 cells were grown and subject to membrane-polysome fractionation. A total of 6 samples, (3 replicates of 2 fractions) were included in the experiment

2010-04-07 | E-GEOD-14037 | biostudies-arrayexpress

Membrane-polysome associated fractionation of HES2 embryonic stem cells

2009-10-01 | GSE14037 | GEO

Translatome of aged mouse NEBD – stage oocytes determined using a new polysomal profiling method

Project description:We provide complete translatome of mouse oocytes from aged females after the breakdown of nuclear envelope (NEBD). RNAseq of polysomal and non-polysomal RNA was determined by using a novel approach combining polysomal fractionation of 200 NEBD-staged oocytes in small SW55Ti centrifugation tubes in the absence of any external spike and qRT-PCR analysis of ribosomal RNA in collected fractions. We compared these data to the translatome of same stage oocytes from young females (GSE121358). We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show the majority of aberrantly translated mRNAs in older oocytes have roles associated with cell cycle regulation. Indeed, we demonstrate the differential translation rates of four specific maternal age related transcripts (Sgk1, Castor1, Aire and Eg5) are associated with distinct localization patterns around the newly forming meiotic spindle.

2021-01-01 | GSE151836 | GEO

Identifying the Translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling)

Project description:Identifying the Translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling)

| PRJNA849932 | ENA

Polysome profiling of Arabidopsis translatome upon hypertonic stress

Project description:Polysome profiling of Arabidopsis translatome upon hypertonic stress

| PRJNA798023 | ENA

Regulation of highly expressed hCINAP on translatome

Project description:hCINAP is essential for human 18S rRNA processing and ribosome assembly. hCINAP is highly expressed in human cancers and promotes cancer cell growth. To connect the role of hCINAP in ribosome biogenesis and tumor growth, genome-wide polysome profiling was performed to detect the regulation of hCINAP on ribosome assembly and translation control. The results showed taht hihgly expressed hCINAP promotes ribosome biogenesis and selectively modulates cancer-associated translatome to promote malignancy. This result provides important insights into the molecular mechanism underlying the role of hCINAP in tumorigenesis. For the transcriptional profile, MCF7 cells transfected with control vector or GFP-hCINAP were collected and total RNA was extracted. For the polysoem profile, MCF7 cells with stably expressed GFP or GFP-hCINAP were subjected to surcose gradient fractionation. Ribosome profiles were obtained by measuring the absorbance of the sucrose gradient at a wavelength of 254 nm using a BioComp Gradient Fractionator. The fractions corresponding to subpolysome and polysome were collected and RNA was extracted. The cDNA library was generated and sequenced via Illumina HiSeqTM 2000. The clean reads were mapped to the UCSC hg19 reference genome and FPKM was assigned to calculate expression levels. The recruitment to polysome of each mRNA was calculated according to their FPKM values in the monsome and polysome.

2016-07-18 | E-GEOD-81469 | biostudies-arrayexpress

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