Mechanism Of Phase Separation In MRNA Degradation And Oocyte Development

Liquid-liquid Phase separation(LLPS)plays an important role in a variety of biological processes,and related with the diseases which caused by protein abnormal aggregation.Previous studies showed that phase separation is significantly on the formation of membraneless organelles.Such as the formation of stress granule,huge transcription factors complex,balbiani body,regulating the storage of maternal RNA in the period of oocyte development,and controlling the assembly of germ granules to determine which embryonic cells develop into germ cells.In this study,we focused on the mechanism of phase separation in mRNA degradation and oocyte development.Part one:YTHDF1 regulates mRNA degradation through phase separationObjectives:N~6-methyladenosine(m~6A)is the most prevalent internal messenger RNA(mRNA)modification and regulates stability of targeting mRNAs.The m~6A reader protein mainly group into the YTH-domain-containing proteins(YTHDF1/2/3 and YTHDC1/2)and IGF2BP1/2/3.Mounting evidences have shown that liquid-liquid phase separation(LLPS)underlies the formation of membraneless structures,and the mechanism of phase separation regulating mRNA degradation remains unclear.In our study,we found that deletion of YTHDF1 caused RNA patchs deposited in He La cells.Our study aims to explore the molecular mechanism of RNA patchs deposition caused by YTHDF1 deletion.Materials and Methods:CRISPR/Cas9 knock out system was used to establish the YTHDF1 knock out cell lines(HEK293 and He La)and METTL14 knock out cell line(HEK293).4SU-TT-seq was used to check the half-life changes of mRNAs.Actinomycin D and q PCR were used to test the half-life changes of individual mRNA.Co-localization of YTHDF1 and AGO2 was identified by immunofluorescence.The interaction domain of YTHDF1 and AGO2 was identified by western blot.Phase separation of YTHDF1 was performed in vitro and in vivo,FRAP was performed on droplets as an assessment of their liquidity.Results:The results of 4SU-TT-seq showed that deletion of YTHDF1 would prolong the half-life of mRNAs.Immunofluorescence data showed that YTHDF1 and AGO2could localize in P-body,and Co-IP results showed that YTHDF1 could interact with AGO2 through YTH domain.We confirmed that YTHDF1 could undergo phase separation in vitro and in vivo,and rather than AGO2,YTHDF1 was the leading factor in P-body formation.The FRAP results showed that liquid AGO2 droplets would convert to gel/solid when YTHDF1 was deleted.Conclusions:We found that the m~6A reader YTHDF1 recruits AGO2(AGO2 perhaps along with mi RNA)through the YTH domain.YTHDF1 degrades targeting mRNAs by promoting P-body formation through LLPS.The deletion of YTHDF1 change the P-body from liquid droplets to gel droplets,and form AGO2/RNA patches,resulting in a degradation delay of mRNAs.Our finding show that YTHDF1 can regulate mRNA degradation through phase separation,and provides a new perspective on the mechanism of the m~6A regulation.Part two:PSPC1 regulates CHK1 phosphorylation and mouse oocyte maturation through phase separationObjectives:Oocytes maturation refers to the process of primary oocytes breaking through the GV phase,generating germinal vesicle breakdown(GVBD),and developing from the first meiotic division to the middle stage of the meiotic division II.A large number of physiological processes are involved in the maturation of oocytes,and the regulation mechanism is extremely complicated.PSPC1 is a paranuclear spot protein,it was reported to function in the occurrence and development of tumors,but the relationship between PSPC1 and oocyte maturation remains unknown.Our study aims to explore the effects of PSPC1 in mouse oocytes maturation.Materials and Methods:Phase separation of PSPC1 was performed in vitro and in vivo,FRAP was performed on droplets as an assessment of their liquidity.Microinjection and si RNA were used to detect the effects of PSPC1 in mouse oocyte maturation.Co-IP and western blot was used to detect the protein,which would interact with PSPC1.Co-localization was identified by immunofluorescence.Results:We confirmed that YTHDF1 could undergo phase separation in vitro and in vivo and the phase separation would be affected by protein concentration,temperature and salt concentration.Pr LD domain was necessary for PSPC1 phase separation and deletion of this domain would significantly weaken the phase separation of PSPC1.We found that knock down expression of PSPC1 would inhibit the mouse oocytes maturation in vitro.Immunofluorescence of spindle showed that oocytes development would arrest at MI when PSPC1 was knocked down.Co-IP results showed that PSPC1would interact with PPP5C and regulated the phosphorylation of CHK1,Pr LD domain of PSPC1 was necessary in this process.Conclusions:In this study,we found that PSPC1 could undergo phase separation and down-regulate CHK1 phosphorylation through PPP5C.In this process,deprivation of the phase separation ability of PSPC1 will lead to a failure of its regulatary function.Down-regulating the expression of PSPC1 by si RNA would greatly inhibit the maturation of mouse oocytes in vitro.Our research reveals that PSPC1 can regulate the CHK1 phosphorylation through phase separation,and play an important role in the maturation of mouse oocytes,providing a new insight into the maturation mechanism of oocytes.