O-13: Phosphorylation of 4E-BP1 Promotes Translation at The Oocyte Spindle

Background: Fully grown mammalian oocyte utilizes transcripts synthetized and stored during earlier development. In the mouse oocyte there are three forms of cap-dependent translational repressors: 4E-BP1, 4E-BP2, and 4E-BP3. The dominant form, 4E-BP1, inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP1 disrupts this inhibitory interaction and results in activation of cap-dependent translation. Materials and Methods: We used immunofluorescence, immunoblotting, qPCR, in situ translation labeling, and microinjection techniques. Results: 4E-BP1 is highly phosphorylated after NEBD, while it is dephosphorylated after fertilization. Increased phosphorylation of 4E-BP1 (which is not detected in the cumulus cells) promotes capdependent translation of specific mRNAs after meiotic resumption. Our immunofluorescence analyses of the differently phosphorylated forms of 4E-BP1 in the oocytes during meiosis show even localization of 4EBP1 and phospho-4E-BP1(T37/46) as well as spindle poles localization of phospho-4E-BP1(S65). 4E-BP1 phosphorylated on T70 co-localizes with its activator mTOR exclusively at the spindle. In addition, mTOR, and CDK1 are the main positive regulators of 4E-BP1 phosphorylation after NEBD; on the other hand, inhibition of PLK1 does not affect 4E-BP1 phosphorylation. Treatment by Rapamycin, inhibitor of mTOR, results in decreased phosphorylation of 4E-BP1 on T37/46 in the whole oocyte, while T70 phosphorylation is decreased at the spindle. Conclusion: Our results show that 4E-BP1 phosphorylation forms promote in situ translation necessary to support spindle assembly and genomic stability.