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.

• 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

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

• Klíčová slova
• 3′RACE, 4EHP, Alternative polyadenylation, Polyadenylation signal, Translation, eIF4E, eIF4E2, eIF4E3,
• MeSH
• 3' nepřekládaná oblast MeSH
• buněčné linie MeSH
• eukaryotický iniciační faktor 4E genetika   metabolismus   MeSH
• exprimované sekvenční adresy MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mozek metabolismus   MeSH
• placenta metabolismus   MeSH
• polyadenylace genetika   MeSH
• proteiny vázající čepičku mRNA genetika   MeSH
• regulace genové exprese MeSH
• sestřih RNA genetika   MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• EIF4E2 protein, human MeSH Prohlížeč
• eIF4E3 protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 4E MeSH
• messenger RNA MeSH
• proteiny vázající čepičku mRNA MeSH