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Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling. A Novel Polysome Fractionation Method
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SYSNO ASEP 0523873 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling. A Novel Polysome Fractionation Method Author(s) Mašek, T. (CZ)
del Llano, Edgar (UZFG-Y) ORCID
Gahurová, Lenka (UZFG-Y) ORCID
Kubelka, Michal (UZFG-Y) RID, ORCID
Šušor, Andrej (UZFG-Y) RID, ORCID
Roučová, K. (CZ)
Lin, C. J. (GB)
Bruce, A. W. (CZ)
Pospíšek, M. (CZ)Article number 1254 Source Title International Journal of Molecular Sciences. - : MDPI
Roč. 21, č. 4 (2020)Number of pages 23 s. Publication form Online - E Language eng - English Country CH - Switzerland Keywords polysome profiling ; polysome fractionation ; translatome ; mouse oocyte ; mouse zygote Subject RIV EB - Genetics ; Molecular Biology OECD category Developmental biology R&D Projects GA19-13491S GA ČR - Czech Science Foundation (CSF) Method of publishing Open access Institutional support UZFG-Y - RVO:67985904 UT WOS 000522524400081 EID SCOPUS 85079586112 DOI 10.3390/ijms21041254 Annotation 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. Workplace Institute of Animal Physiology and Genetics Contact Jana Zásmětová, knihovna@iapg.cas.cz, Tel.: 315 639 554 Year of Publishing 2021 Electronic address https://www.mdpi.com/1422-0067/21/4/1254
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