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Open AccessArticle

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

1
Laboratory of RNA Biochemistry, Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
2
Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal Physiology and Genetics, CAS, Rumburská 89, 277 21 Liběchov, Czech Republic
3
Laboratory of Early Mammalian Developmental Biology (LEMDB), Department of Molecular Biology and Genetics, Faculty of Science, University of South Bohemia, 370 05 České Budějovice, Czech Republic
4
Queen’s Medical Research Institute, MRC Centre for Reproductive Health, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(4), 1254; https://doi.org/10.3390/ijms21041254
Received: 7 January 2020 / Revised: 3 February 2020 / Accepted: 10 February 2020 / Published: 13 February 2020
(This article belongs to the Special Issue Translational Control 2.0)
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. View Full-Text
Keywords: polysome profiling; polysome fractionation; translatome; mouse oocyte; mouse zygote; mouse early embryo; SW55Ti rotor; RNA-seq polysome profiling; polysome fractionation; translatome; mouse oocyte; mouse zygote; mouse early embryo; SW55Ti rotor; RNA-seq
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MDPI and ACS Style

Masek, T.; del Llano, E.; Gahurova, L.; Kubelka, M.; Susor, A.; Roucova, K.; Lin, C.-J.; Bruce, A.W.; Pospisek, M. Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method. Int. J. Mol. Sci. 2020, 21, 1254.

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