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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE151836 | GEO | 2021/01/01

REPOSITORIES: GEO

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Project description:Translatome analysis by sucrose gradient centrifugation of cell lysates followed by microarray profiling of the polysomal and subpolysomal RNA fractions represents a way of both studying translational control networks and better approximating the proteomic representation of cells. It is an established notion that translational control takes place essentially at the translation initiation level, therefore the variation in abundance of a given mRNA species on polysomes can be directly related to the variation in abundance of the corresponding protein. Comparison of translatome profile changes with corresponding transcriptome profile changes can provide a measure of the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. To provide a direct experimental evaluation of the phenomenon, we decided to study a classical example of transcriptional reprogramming of gene expression: Epidermal Growth Factor (EGF) treatment. This stimulus triggers a well known chain of intracellular transduction events, ultimately resulting in a multifaceted phenotypic spectrum of changes with prevalent induction of cell growth and proliferation. We subjected HeLa cells to serum starvation for 12h and then we added EGF at final concentration of 1 Î¼g/ml, profiling before and after 40 minutes of treatment the transcriptome, the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation, and also the mRNA content of the subpolysomal pool, expected not to be actively translated. Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, EGF starvation release. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved HeLa cells transcriptome in response to EGF stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA, subpolysomal RNA and total RNA were isolated from HeLa cells serum starved and treated with EGF. Cells lysates were collected before (t = 0 min) and after (t = 40 min) EGF treatment. All experiments were run in triplicates.

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

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