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

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 q...

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Autores principales: Masek, Tomas, del Llano, Edgar, Gahurova, Lenka, Kubelka, Michal, Susor, Andrej, Roucova, Kristina, Lin, Chih-Jen, Bruce, Alexander W., Pospisek, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072993/
https://www.ncbi.nlm.nih.gov/pubmed/32070012
http://dx.doi.org/10.3390/ijms21041254
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author Masek, Tomas
del Llano, Edgar
Gahurova, Lenka
Kubelka, Michal
Susor, Andrej
Roucova, Kristina
Lin, Chih-Jen
Bruce, Alexander W.
Pospisek, Martin
author_facet Masek, Tomas
del Llano, Edgar
Gahurova, Lenka
Kubelka, Michal
Susor, Andrej
Roucova, Kristina
Lin, Chih-Jen
Bruce, Alexander W.
Pospisek, Martin
author_sort Masek, Tomas
collection PubMed
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.
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spelling pubmed-70729932020-03-19 Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method Masek, Tomas del Llano, Edgar Gahurova, Lenka Kubelka, Michal Susor, Andrej Roucova, Kristina Lin, Chih-Jen Bruce, Alexander W. Pospisek, Martin Int J Mol Sci Article 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. MDPI 2020-02-13 /pmc/articles/PMC7072993/ /pubmed/32070012 http://dx.doi.org/10.3390/ijms21041254 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Masek, Tomas
del Llano, Edgar
Gahurova, Lenka
Kubelka, Michal
Susor, Andrej
Roucova, Kristina
Lin, Chih-Jen
Bruce, Alexander W.
Pospisek, Martin
Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title_full Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title_fullStr Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title_full_unstemmed Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title_short Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
title_sort identifying the translatome of mouse nebd-stage oocytes via ssp-profiling; a novel polysome fractionation method
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072993/
https://www.ncbi.nlm.nih.gov/pubmed/32070012
http://dx.doi.org/10.3390/ijms21041254
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