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

0523873 - ÚŽFG 2021 RIV CH eng J - Journal Article
Mašek, T. - del Llano, Edgar - Gahurová, Lenka - Kubelka, Michal - Šušor, Andrej - Roučová, K. - Lin, C. J. - Bruce, A. W. - Pospíšek, M.
Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling. A Novel Polysome Fractionation Method.
International Journal of Molecular Sciences. Roč. 21, č. 4 (2020), č. článku 1254. E-ISSN 1422-0067
R&D Projects: GA ČR(CZ) GA19-13491S
Institutional support: RVO:67985904
Keywords : polysome profiling * polysome fractionation * translatome * mouse oocyte * mouse zygote
OECD category: Developmental biology
Impact factor: 5.924, year: 2020
Method of publishing: Open access
https://www.mdpi.com/1422-0067/21/4/1254

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.
Permanent Link: http://hdl.handle.net/11104/0308150