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.

• Keywords
• RNA-seq, SW55Ti rotor, mouse early embryo, mouse oocyte, mouse zygote, polysome fractionation, polysome profiling, translatome,
• MeSH
• Nuclear Envelope genetics   metabolism   MeSH
• Meiosis genetics   MeSH
• Mice MeSH
• Oocytes growth & development   metabolism   MeSH
• Polyribosomes genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• RNA, Messenger, Stored genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• RNA, Ribosomal, 28S genetics   MeSH
• RNA-Seq MeSH
• Gene Expression Regulation, Developmental genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA-Binding Proteins MeSH
• RNA, Messenger, Stored MeSH
• RNA, Ribosomal, 18S MeSH
• RNA, Ribosomal, 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.

• Keywords
• 3′RACE, 4EHP, Alternative polyadenylation, Polyadenylation signal, Translation, eIF4E, eIF4E2, eIF4E3,
• MeSH
• 3' Untranslated Regions MeSH
• Cell Line MeSH
• Eukaryotic Initiation Factor-4E genetics   metabolism   MeSH
• Expressed Sequence Tags MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Brain metabolism   MeSH
• Placenta metabolism   MeSH
• Polyadenylation genetics   MeSH
• RNA Cap-Binding Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• RNA Splicing genetics   MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3' Untranslated Regions MeSH
• EIF4E2 protein, human MeSH Browser
• eIF4E3 protein, human MeSH Browser
• Eukaryotic Initiation Factor-4E MeSH
• RNA, Messenger MeSH
• RNA Cap-Binding Proteins MeSH