Interleukin-1α (IL-1α) is a dual-function proinflammatory mediator. In addition to its role in the canonical IL-1 signaling pathway, which employs membrane-bound receptors, a growing body of evidence shows that IL-1α has some additional intracellular functions. We identified the interaction of IL-1α with the tumor suppressor p53 in the nuclei and cytoplasm of both malignant and noncancerous mammalian cell lines using immunoprecipitation and the in situ proximity ligation assay (PLA). This interaction was enhanced by treatment with the antineoplastic drug etoposide, which suggests a role for the IL-1α•p53 interaction in genotoxic stress.

• MeSH
• Cytoplasm metabolism   MeSH
• DNA Breaks, Double-Stranded MeSH
• Microscopy, Fluorescence MeSH
• HeLa Cells MeSH
• Immunoprecipitation MeSH
• Interleukin-1alpha genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• DNA Damage genetics   physiology   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-1alpha MeSH
• Tumor Suppressor Protein p53 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.

• 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

BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. RESULTS: We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. CONCLUSION: Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.

• Keywords
• Arsenite, Eukaryotic translation initiation factor 4E (eIF4E), Heat shock, PB, Processing body (P-body), SG, Stress granule, Translation control, Translation initiation factor, eIF4E2, eIF4E3,
• MeSH
• Cell Line MeSH
• Cytosol metabolism   MeSH
• Eukaryotic Initiation Factor-4E analysis   genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Oxidative Stress * MeSH
• Poly(A)-Binding Protein I analysis   metabolism   MeSH
• RNA Cap-Binding Proteins analysis   genetics   metabolism   MeSH
• Heat-Shock Response * MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EIF4E2 protein, human MeSH Browser
• eIF4E3 protein, human MeSH Browser
• Eukaryotic Initiation Factor-4E MeSH
• RNA, Messenger MeSH
• Poly(A)-Binding Protein I MeSH
• RNA Cap-Binding Proteins MeSH