The Function Research And Mechanism Exploration Of M6A Recognition Protein Ythdc2 In Gametogenesis

BackgroundThe classic epigenetic modification mainly focuses on DNA methylation(5mC)and histone modifications(acetylation and phosphorylation),while there are few researches about the RNA methylation at present.In recent years,a large number of achievements has been obtained.These discoveries has opened a new realm for post-transcriptional gene regulation.N6-methyladenosine(m6A)is identified as the most abundant internal modification of messenger RNA in eukaryotes and it plays an important role in the development of biology.Proteins mediating the effects of m6A establish an interplay among m6A deposition,removal and recognition factors("writers","erasers" and "readers",respectively).The biological functional investigations of m6A-related modification mainly focus on its recognition protein.Spermatogenesis is a progress characterized by the dynamic changes of gene expression and syntheses and degradations of RNA and RNA recognition proteins.Thus it is an ideal model for RNA recognition protein studies.It still remains elusive about how the m6A recognition protein,as a classification of RNA binding proteins,works on the spermatogenesis.We speculate that Ythdc2 was also a specific m6A recognition protein and it can specifically recognize the m6A through its "YTH" domain like YTHDF1-3 and YTHDC1.Therefore,we picked up the Ythdc2 protein,as the targeted protein in our research.We tried to find out its potential functions in the spermatogenesis(such as the mitotic phase,meiosis and spermatogenesis by gene knockout mice models.Thus,we could further explore the biological functions about m6A-related modification by studying the recognition proteins.Methods and resultsAdvantage expression of Ythdc2 gene in the brain and testicular tissue in mice was conformed by RT-PCR,which suggested the potential effects of Ythdc2 on the nervous system and reproductive system developmentUsing CRISPR/Cas9 technology combined with pronucleus microinjection,the knockout mice model of Ythdc2(lack of 37 bp and 4 bp respectively)was generated.In this study we choose the former model for further research.Both female and male of Ythdc2 knockout mice were infertile.The male mice showed smaller testes compared with the wide-type ones.The spread of spermatocyte confirmed that the Ythdc2-deficient mice failed to finish meiosis and it arrested at the zygotene stage of the first meiotic prophase.In addition,the testicular luminal cells appeared apoptosis firstly at postnatal 9.5 d;for female mice,the meiosis progress also arrested and the primitive follicles appeared apoptosis after birth.The formation of synaptonemal complex(SC)was abnormal.In addition,samples of testes at E8.5 d mice were used for high throughput sequencing.Compared with wild type,the genes(Sycpl,Sycp2,Stag3,Smc1?,etc.)involved in meiosis were downregulated.Therefore,the deletion of Ythdc2 resulted in the abnormal formation of synaptonemal complex for both male and female mice.ConclusionAbove all,we speculated that,in the deficiency of Ythdc2,the stability of the mRNA was affected.For the reproductive system,both female and male were arrested at meiosis prophase I(MPI),and the germ cells appeared apoptosis.