Studies On The Function Of Smg7 In Mouse Embryonic Stem Cells And DNA Damage Repair

Nonsense-mediated mRNA decay(NMD)is a well-conserved post-transcriptional regulation mechanism in eukaryotic cells.NMD is not only responsible for degrading mRNA with a premature termination codon(PTC),but also degrades a part of mRNA which could be translated into functional proteins.Thus,NMD is an RNA quality and quantity surveillance mechanism.Two branches of mRNA degradation are illustrated in NMD.SMG6 has the endonuclease activity,which can cleave target mRNA at the vicinity of PTC.SMG5 and SMG7 form heterodimer,and recruits DCP1 and CCR-NOT4 to facilitate exonuclease mediating mRNA degradation.Besides,it has been suggested that SMG7 could regulate p53 stability.Thus,SMG7 could participate in NMD degradation and DNA damage repair response.In this study,we constructed Smg7 conditionally knockout mice by conditional gene targeting,and created Smg7 induced knockout mice by mating with Cre-ERT2+mice.Based on this,we successfully isolated and established Smg7 induced mouse embryonic stem cell lines(mESCs).Below are the research topics and results discussed:(1)We have successfully generated Smg7 knockout mESCs by 4-OHT induction in Smg7F/FCre-ERT2+mESCs.Our results showed that other core NMD factors,such as Smg5,Smg6 and Upfl,were significantly increased on protein level and mRNA level in Smg7 knockout mESCs,but the compensatory increase of these NMD factors did not rescue the inhibition of NMD in Smg7 knockout mESCs.In addition,in Smg7 knockout mESCs,PTC+mRNAs transcripts were significantly accumulated,and classical non-PTC dependent NMD targets,such as Atf4,Ddit3,Gas5,Snhgl2 were also significantly increased on mRNA level.These results indicate that Smg7 is a major NMD factor in mESCs,and loss of Smg7 results in full NMD inhibition of mESCs.(2)We previously showed that knock-down or knockout of NMD factors,such as Smg5 and Smg6,caused mESCs differentiation defect.Here,by employing embyroid body formation assay and spontaneous differentiation assay,we investigated differentiation capacity of Smg7 knockout mESCs.We found,as compared with control mESCs,Smg7 knockout mESCs has severe differentiation defects.qPCR and Western blotting analysis show that stem cell transcription factors Oct4A,Sox2 and c-Myc are highly represented in Smg7 knockout mESCs.These findings strongly indicated that Smg7 is a regulatory factor of cell fates in mESCs,which also reinforced the hypothesis that"NMD licenses cell fates of mESCs differentiation".(3)In the human cancer cells(HCT116),Smg7 regulates p53 stability upon DNA damage.In order to explore the function of Smg7 in DNA damage repair of mESCs,we used Doxorubicin(Dox)at a concentration of 500ng/mL to induce DNA damage in mESCs.Western blotting analysis on p53 protein level showed no difference between control and Smg7 null mESCs.Otherwise,qPCR analysis on mRNA levels of p53 and p21 showed some changes between control and Smg7 knockout mESCs under normal condition while showed no changes being treated by Dox.These findings clearly indicate that,at least in mESCs,Smg7 does not regulates p53 signal channel under DNA damage.To sum up,in current researchs,by constructing Smg7 conditional knockout mouse and establishing Smg7 knockout embryonic stem cell line,we investigated the roles of Smg7 in mESCs biology.Current findings strongly reinforced the working hypothesis that NMD regulates cell fates transition of mESCs self-renewal and differentiation.The mouse and cell models generated will be valuable tools for future studies on elucidating NMD biology.