The APC/C Complex Coordinates With OMA-1 To Regulate The Oocyte-to-embryo Transition In C.elegans

During oocyte maturation and the oocyte-to-embryo transition,key developmental regulators such as RNA-binding proteins coordinate translation of particular mRNA and related developmental processes by binding to their cognate maternal mRNAs.In Caenorhabditis elegans(C.elegans),oocyte-to-embryo transition and early embryogenesis are rapid and dynamic,which is a classical model system for studying the gamete-zygote transition process.In C.elegans,these processes are regulated by a set of CCCH zinc finger proteins.Oocyte maturation defective-1(OMA-1)and Oocyte maturation defective-2(OMA-2)are two functionally redundant CCCH zinc finger proteins.OMA-1/2 binds to target mRNA and inhibits its translation during oocyte maturation;During the oocyte-to-embryo transition,OMA-1/2 protein degrades rapidly,releasing target mRNA,which begins translation.Moreover,OMA-1/2 interacts with the transcription factor TATA Box Binding Protein Associated Factor-4(TAF-4)in early embryos and binds TAF-4 to the cytoplasm,inhibiting the transcription of embryos.Phosphorylation of OMA-1 promotes degradation of OMA-1 and releases TAF-4 into the nucleus to initiate transcription in embryonic cells.Therefore,rapid metabolism of OMA-1/2 during the first embryonic cell division is necessary for proper transition from oogenesis to embryogenesis.Although the events of OMA-1/2 mediated oocyte maturation and oocyte-to-embryo transition have been reported,but the detailed mechanism and regulation of this process remain unclear and need to be In-depth research.OMA-1 is located in the cytoplasm during oocyte maturation and in the germ granules of C.elegans,also known as P granules,in early embryos,and performs transcriptional inhibition.Germ granules are phase separation droplets that exist in germ cells of all living organisms and contain non-coding RNA mRNA and various proteins needed for germline development,with the function of maintaining the fate of germ cells.In the development of the reproductive system,both the localization and formation of P granules are highly dynamic,and the specific regulatory mechanisms involved in this process are not very clear up to now.Objection: To study the role and regulatory mechanism of RNA-binding protein OMA-1/2 in oocyte maturation and the oocyte-to-embryo transition of C.elegans;To study the formation and localization mechanism of P granules.Methods: This project mainly utilizes genetics,cell biology,biochemistry,molecular biology,transcriptome approaches to study the subject.Results: In the first part of the study,a total of 26 mutant strains were isolated by forward genetic screening of oma-1(zu405),and the identified candidate mutant genes all encode the subunits of APC/C complex.Knockdown of these genes inhibited the lethal phenotype of oma-1(zu405),reconfiguration of these mutated genes using CRISPR/Cas9 gene editing techniques also inhibited the lethal of oma-1(zu405),suggesting that the encoding gene of APC/C complex subunit was the functional gene.Transcriptome analysis showed that mutations in the subunit of the APC/C complex corrected the disordered early embryonic transcriptome of oma-1(zu405)and partially restored the wild-type N2 transcriptome phenotype.In addition,the binding of OMA-1 to mRNA was affected,resulting in the type of mRNA binding to OMA-1 in oma-1(zu405)was similar to that of wild type N2.Fluorescence quantification and localization analysis of embryos showed that the APC/C complex subunit mutation did not affect the expression of OMA-1 in the transgenic strains,but affected the distribution of OMA-1 in the transgenic strains in early embryos,and significantly reduced the localization of P granules in 4-cell embryos,and the abnormal distribution of cell fate determinants in oma-1(zu405)was corrected to resemble the wild-type N2 phenotype.In the second part,it was found that the formation and localization of P granules were regulated by Germ Line Helicase-1(GLH-1),a VASAlike RNA Helicase.GLH-1 couples distinct steps of its ATPase hydrolysis cycle to control the formation and disassembly of P granules;and the phenylalanine-glycineglycine(FGG)repeats in GLH-1 are required for its perinuclear localization and perinuclear localization with P granules components.Moreover,the loss of P granules component also affected the fertility of C.elegans.Conclusion: During oocyte maturation and the oocyte-to-embryo transition of C.elegans,the APC/C complex has a genetic relationship linking with OMA-1.we found that mutations in the genes encoding the subunit of the APC/C complex inhibited embryonic lethality of the strains of oma-1(zu405).Mutations of the APC/C subunits alter gene expression in early embryos of oma-1(zu405)mutant to wild-type N2 and correct the abnormal distribution of some key cell fate determinants.Further studies showed that,although the APC/C complex does not regulate the degradation of OMA-1,it promotes P granule accumulation and modifies mRNA binding of OMA-1 to regulate the oocyte-to-embryo transition process.The formation and localization of P granules were regulated by RNA helicase GLH-1.Different domains of GLH-1 cooperate with each other to strictly regulates the dynamics and localization of P granules and substance exchange with cell pool,which plays versatile role in controlling the formation of phase separation droplets in space and time to ensure the normal development of organisms.