Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method
Jazyk angličtina Země Švýcarsko Médium electronic
Typ dokumentu časopisecké články
Grantová podpora
MR/N022556/1
Medical Research Council - United Kingdom
19-13491S
Grantová Agentura České Republiky
PubMed
32070012
PubMed Central
PMC7072993
DOI
10.3390/ijms21041254
PII: ijms21041254
Knihovny.cz E-zdroje
- Klíčová slova
- RNA-seq, SW55Ti rotor, mouse early embryo, mouse oocyte, mouse zygote, polysome fractionation, polysome profiling, translatome,
- MeSH
- jaderný obal genetika metabolismus MeSH
- meióza genetika MeSH
- myši MeSH
- oocyty růst a vývoj metabolismus MeSH
- polyribozomy genetika MeSH
- proteiny vázající RNA genetika MeSH
- RNA messenger skladovaná genetika MeSH
- RNA ribozomální 18S genetika MeSH
- RNA ribozomální 28S genetika MeSH
- sekvenování transkriptomu MeSH
- vývojová regulace genové exprese genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny vázající RNA MeSH
- RNA messenger skladovaná MeSH
- RNA ribozomální 18S MeSH
- RNA ribozomální 28S MeSH
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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