PGC7 Promotes Maternal MRNA Translation In Mouse Oocytes By Regulating The Substrate Specificity Of AKT Kinase For YBX1

The production of mammalian oocytes has gone through a long process of oogenesis,and the fully developed oocytes are blocked in the two-line phase of the first meiosis.At this time,a large amount of maternal mRNA is stored in the oocyte,and maternal gene transcription is in silence.The early developmental processes,including meiotic spindle assembly,maintenance of MⅡ arrest,and mRNA clearance during maternal-to-zygotic transition（MZT）,are completely driven by the maternal protein and maternal mRNA translation.The timely and accurate translation of maternal mRNA provides important material guarantee for oocyte maturation and early embryo development.mRNA 7-methylguanylate（m⁷G）cap structure and polyadenylation（poly A）tail are involved in the activation translation.Most（80%）mRNAs in mouse oocytes have m⁷G cap structure.The translation initiation factor eIF4E that recognize the m⁷G cap structure is regulated by phosphorylation of a variety of upstream kinases and participate in the regulation of the translational activation of maternal mRNA.AKT（also known as PKB）is an important serine/threonine protein kinase that cooperates with other kinases such as ERK1/2 to regulate oocyte maturation and early embryo development.The translation of partial maternal mRNA is regulated by the mTOR-eIF4F pathway,which is located at downstream of AKT.At the same time,the activation of phosphorylation of AKT has priority over maternal mRNA translation during oocyte maturation,suggesting that AKT is also involved in the process of the activation of maternal mRNA translation,Maternal protein is essential in oocyte maturation and early embryonic development.PGC7 is an important maternal protein.The main function of PGC7 is currently known to inhibit the demethylation of maternal genome by TET3 at the zygote and maintain the stability of DNA methylation of maternal imprinted genes.If the maternal Pgc7 is knocked out,the development of most mouse embryos will be stagnated in the 2-cell stage,and the paternal Pgc7 alleles that are expressed during the 2-cell stage cannot save the developmental block caused by the maternal Pgc7 knockout.Meanwhile,the expression of imprinted genes mainly affects the placenta development of post-implantation embryo,suggesting that an important function of PGC7 has not been recognized in the early development process.This paper revealed the function of maternal PGC7 in early developmental events and obtained the following results.1.PGC7 regulates oocyte maturation and early embryonic development.The results of immunofluorescence staining showed that PGC7 was expressed in both oocytes and fertilized embryos.The expression of PGC7 was the highest in GVBD and showed obvious enrichment around chromosomes.Compared with Pgc7^+/+and Pgc7^+/-,approximately 65%of embryos had undergone fragmentation at the follicular stage in Pgc7^-/-mouse and the maturation rate of Pgc7^-/-mouse was reduced to about 40%in vitro;90%embryos of Pgc7^-/-mouse were stagnated in the 2-cell stage and the remaining 10%could develop to 4-cell,but these partial 4-cell embryos could not continue to develop.Cyclin B1 is a key component of oocyte maturation factor MPF,and its expression level directly determines MPF activity.Western Blot and immunostaining found that the expression of Cyclin B1 protein decreased significantly in the MⅡ oocytes of Pgc7^-/-mouse,but RT-qPCR found that its transcription did not change significantly.The above results indicated that part of the reason why oocyte maturation rate was reduced was that Pgc7 knockout caused the obstruction Cyclin B1 translation.Protein silver staining experiments found that the expression of total protein was significantly reduced in the GVBD-phase oocytes and zygotes of Pgc7^-/-mouse.Newborn protein synthesis efficiency test confirmed that the protein synthesis rate was significantly inhibited by Pgc7 knockout in GVBD oocytes.Cell experiment showed that overexpression of exogenous PGC7 in HEK293T cells significantly promoted global protein translation.These results suggest that PGC7 is directly involved in the translation activation of maternal mRNA.2.PGC7 regulates AKT activity and substrates phosphorylation modificaion of AKT.The signaling pathway of mTOR and ERK1/2 were involved in the activation of maternal mRNA translation.Immunofluorescence staining showed that mTOR and ERK1/2phosphorylation was not changed significantly in Pgc7^+/+,Pgc7^+/-and Pgc7^-/-mouse oocytes.Western Blot and immunofluorescence showed that AKT activity was significantly inhibited in Pgc7^+/-mouse oocytes,while was significantly activated in Pgc7^-/-mouse oocytes.The detection of neonatal protein translation synthesis found that p-AKT-473decreased,but the translation efficiency of neonatal protein did not change significantly in Pgc7^+/-mouse oocytes,while p-AKT-473 was significantly increased,but the translation efficiency of nascent protein decreased significantly in Pgc7^-/-mouse oocytes.It indicated that the increase of p-AKT-473 could not activate the translation of maternal mRNA in the absence of PGC7.Immunofluorescence revealed that p-AKT-Ser473 gradually increased from GV to MⅡ oocytes,reaching the highest peak in MⅡ stage,which was evenly distributed in the oocyte;p-AKT-Ser473 was lower than that in MⅡ stage after fertilization and tended to be stable as the embryo developed.The inhibition of AKT activity by MK2206 led to the obstruction of oocyte maturation.In early embryos,the first cleavage time was delayed after inhibiting AKT activity,while the embryo development was blocked at the 2-cell stage.Both protein silver staining experiment and new protein synthesis efficiency assay found that the expression of maternal protein of oocytes in GVBD and in 2-cell embryos significantly reduced after inhibiting the AKT activity.The accumulation of neonatal proteins in the vicinity of chromosomes in the GVBD oocytes is similar to the distribution characteristics of PGC7 during this period,but the neonatal protein is no longer enriched near the chromosome after treatment with MK2206.In HEK293T cells,the efficiency of nascent protein synthesis also decreased significantly after inhibiting AKT activity.These results suggest that inhibition of AKT activity can down-regulate the synthesis of parent protein.However,p-Ak T-473 was significantly increased in Pgc7^-/-mouse oocytes,suggesting that the phosphorylation ability of AKT to its substrate is reduced by the loss of PGC7.Western Blot and immunofluorescence detected that phosphorylation of the AKT substrate motif p-RXRXXS/T in Pgc7^-/-mouse oocytes was significantly reduced;AKT substrate capture experiment confirmed PGC7 can promote AKT to capture more substrates.The above results indicated that PGC7 not only participated in the regulation of AKT activity,but also played an assisted role in the selection of AKT substrates.3.To further study the mechanism of PGC7 in regulating AKT activity,Co-immunoprecipitation（co-IP）experiments confirmed that PGC7 had a clear interaction with the CTD domain of AKT.Immunofluorescence experiments confirmed that PGC7 and AKT are co-localized in each nucleus of the cytoplasm.Overexpression of wild-type PGC7significantly promoted the perinuclear enrichment of endogenous/exogenous AKT.While nuclear export signal mutant PGC7-44/46AA and nuclear localization signal mutant PGC7-KRR had no such function.Co-immunoprecipitation（co-IP）experiments confirmed that the interaction between PGC7-44/46AA and PGC7-KRR with AKT was stronger than wild-type PGC7.Knocking down Pgc7,the phosphorylation of AKT Thr308 and Ser473 was significantly reduced,while overexpression of Pgc7,the phosphorylation of Thr308 and Ser473 increased significantly.Overexpression of Pgc7 also could antagonize the decline of AKT phosphorylation caused by LY294002 and MK2206.Interestingly,knocking down Pgc7 in F9 cell caused the compensatory tendency of cells to activate AKT,which could be expressed by the down-regulation of Pp2a-ca transcription,up-regulation of Pik3ca transcription and up-regulation of PIK3CA protein expression.At the same time,immunofluorescence detection of oocytes found that knocking out Pgc7 significantly promoted the expression of PIK3CA protein,but it still could not rescue the decrease of AKT phosphorylation caused by the deletion of PGC7 protein.Co-immunoprecipitation（co-IP）experiments further confirmed that PGC7 not only promoted the interaction between PDK1 and AKT,but also inhibited the interaction between PP2A and AKT;Overexpression of Pgc7 could significantly antagonize the decrease in AKT phosphorylation caused by PP2A,which indicated that PGC7 maintained AKT activity at two levels:promoting AKT phosphorylation and inhibiting its dephosphorylation.When the PGC7 protein was reduced,its activation role of AKT at the interaction level was significantly reduced.The up-regulation of PIK3CA could not save the decline of AKT phosphorylation.4 PGC7 promoted AKT-YBX1 interaction and increased YBX1s100 phosphorylation.YBX1 interacts with PGC7,and AKT phosphorylates YBX1s100 to activate translation.Therefore,YBX1 is the downstream factor of PGC7 regulating maternal mRNA translation.The results of immunofluorescence showed that YBX1 in oocytes was evenly distributed in the cytoplasm,and the distribution of YBX1 began to decrease significantly in the nucleus after fertilization.p-YBX1 gradually increased during the maturation of oocytes and reached the highest peak in MⅡ phase,which was the same with p-AKT-473 tendency,indicating that AKT was the upstream kinase that directly phosphorylated YBX1s100 in oocytes.In the zygotes,p-YBX1 was enriched in two pronuclear nucleolar regions and decreased significantly in the cytoplasm.Treatment of oocytes with Fisetin,a specific inhibitor of YBX1s100 phosphorylation,significantly inhibit the maturation of oocytes and the embryos were blocked at the 2-cell.The new-born protein synthesis efficiency test found that protein synthesis was significantly inhibited after oocytes,2-cell embryos,HEK293T,NIH3T3,and F9 were treated with Fisetin.Immunofluorescence and Western Blot showed that the phosphorylation of YBX1s100 was significantly decreased in MⅡ oocytes and zygotes of Pgc7^+/-mouse compared with wild-type mouse,while YBX1s100 further decreased significantly in Pgc7^-/-mouse.Immunofluorescence was almost undetectable,but the expression of YBX1 was could not affect.These results suggest that PGC7 can maintain YBX1 phosphorylation.To further study the mechanism of PGC7 in regulating YBX1 phosphorylation,co-IP experiment found that the CSD domain and CTD domain of YBX1 protein could interact with full-length PGC7.The NTD domain and CTD domain of PGC7 protein could also interact with YBX1,but the binding capacity of PGC7 and the CTD domain of YBX1protein was stronger.Immunofluorescence revealed that PGC7 and YBX1 were co-localized in the cytoplasm.The co-IP experiment confirmed that there were simultaneous interactions between PGC7-AKT-YBX1,and PGC7 promoted the interaction between AKT-YBX1.The overexpression of Pgc7 promoted the phosphorylation of YBX1s100 in F9 cells,and decreased the phosphorylation of YBX1s100 when Pgc7 was knocked down;overexpression of Pgc7 could also save the decreased YBX1s100 phosphorylation caused by AKT inhibitor LY294002.Phosphorylation of YBX1s100 can relieve inhibition of eIF4E.The results of co-IP showed that AKT promoted the phosphorylation of YBX1s100 and inhibited interaction of YBX1-eIF4E;PGC7 promoted YBX1s100 phosphorylation by promoting AKT activity and AKT-YBX1 interaction,and further reduced the YBX1-eIF4E interaction.While the interaction of YBX1 and eIF4E was no longer regulated by AKT and PGC7 after the YBX1s100 was mutated to Ala.In order to show that the PGC7-AKT-YBX1 pathway mainly played a regulatory role at the level of translation,we integrated and analyzed the published RIP-seq data of YBX1and the single-cell transcriptome data of oocytes of Pgc7 knockout and found that only 23%of gene expression in Pgc7^-/-mouse oocytes was significantly changed,and 2295 of 3117genes which were detected were YBX1 target genes in oocytes,accounting for 73.58%of the total.At the same time,1838 of these 2295 target genes were not affected by Pgc7knockout,accounting for 80%of the total.GO analysis found that the function of these1838 genes were focused on regulating the mitotic cell cycle,chromosome segregation,and RNA localization.Maternal YAP1 is a key zygotic genome activation factor.RIP-qPCR confirmed that YBX1 could bind to Yap1 mRNA.After knocking out Pgc7,RT-qPCR found that the transcription of Yap1 did not change significantly in oocytes,but Western Blot and Immunofluorescence revealed that its protein expression was significantly reduced.After using Fisetin to inhibit YBX1s100 phosphorylation in oocytes,immunofluorescence detection revealed that YAP1 expression was significantly reducedIn cell experiments,Western Blot detection found that YAP1 expression decreased significantly as YBX1 and AKT phosphorylation decreased when Pgc7 was knocked down in F9 cells.At the same time,F9 cells were treated with Fisetin or MK2206 to inhibit the phosphorylation of YBX1 or AKT,Western Blot detection revealed that the expression of YAP1 was also significantly inhibited.These results suggest that the inhibition of YAP1translation is partly responsible for the 2-cell development retardation caused by Pgc7knockout.5.PGC7 maintains the pluripotency of F9 cells by regulating the expression of Nanog.RT-qPCR and Western Blot showed that Pgc7,Nanog,Sox2,and Oct4 were significantly down-regulated at the level of transcription and protein expression during the differentiation of F9 cells induced by retinoic acid（RA）.However,when the exogenous PGC7 was overexpressed,the transcription of Nanog and down-regulation of the translation of protein induced by RA were suppressed.NANOG protein expression was up-regulated when PGC7was overexpressed,but the transcription level did not change significantly.After knocking down Pgc7 in F9 cells,RT-qPCR and Western Blot showed that the expression of Nanog,Sox2 and Oct4 was down-regulated.The phosphorylation of YBX1s100 weakens its ability to bind to mRNA.The RIP-qPCR confirmed that YBX1 could directly bind to Nanog mRNA.IGF treatment to activate AKT or overexpression of PGC7 could significantly increase the phosphorylation of YBX1s100,which led to the weakening of the binding capacity of YBX1 to Nanog mRNA.Simultaneous stimulation with EGF or IGF for 30-45 minutes in F9 cells,the phosphorylation modification of AKT and YBX1 were the highest,and the expression of NANOG protein was also the highest during this period,but its transcription did not change significantly.On the other hand,After F9 cells were treated with Fisetin or MK2206respectively to inhibit the phosphorylation of YBX1 or AKT,Western Blot analysis showed that the expression of NANOG was significantly inhibited.The above results indicated that the AKT-mediated phosphorylation of YBX1s100 promoted by PGC7 could promote Nanog translation,thereby maintaining the pluripotency of F9 cells.In summary,this study confirms that AKT activation is required to activite the translation of maternal mRNA.PGC7 regulates AKT activity by inhibiting the dephosphorylation of AKT by PP2A and assisting the phosphorylation of specific substrates of AKT.PGC7 was shown to be involved in the translation activation of maternal mRNA by promoting AKT targeted phosphorylation of maternal translation inhibitor YBX1 through protein interaction.PGC7 regulating YBX1 phosphorylation promoted the translation of Cyclin B1,Yap1,and Nanog respectively,which affected oocyte maturation,2-cell developmental and F9 cell pluripotency.