Systematic Analysis Of Expressed Profiles Of LncRNA And CircRNA In Germline Stem Cell And Dosage Compensation In The Process During Their Development

Germline stem cells belong to adult stem cells,and they possess the ability that transmit genetic information from generation to generation.There are two type of germline stem cells,including spermatogonial stem cell(SSC)and female germline stem cell(FGSC).The selfrenewal and differentiation of germline stem cells and the gene dosage balance in the process during their development are of great significance for the reproduction of the organism.Accumulating evidence indicates that long noncoding RNAs(lnc RNAs)and circular RNAs(circ RNAs)involve in germ cell development.However,little is known about the functions and mechanisms of lnc RNAs and circ RNAs in self-renewal and differentiation of germline stem cells.Currently,we systematic identificated and comparied the expression patterns of lnc RNAs and circ RNAs with associated co-expression and ce RNA networks in mouse germline stem cells by high-throughput sequencing.We identified 18573 novel lnc RNAs and 18822 circ RNAs in the germline stem cells and further confirmed the existence of these lnc RNAs and circ RNAs by RTPCR.The number of exon,average length of transcriptions and ORF,and protein coding potential of the novel lnc RNA were similar to the known lnc RNAs.However,the average size of the open reading frames(ORFs)in the lnc RNAs and m RNAs was 86.24 bp and 394.84 bp,respectively,which indicated that the m RNA ORFs were significantly longer than lnc RNAs ORFs.Moreover,the lnc RNA and m RNA transcripts were found to be distributed on all of the mouse chromosomes.Among the novel lnc RNAs in our study,the intergenic lnc RNAs constituted the majority,and the numbers of lnc RNAs of each type were similar between SSCs and FGSCs.Most of the 18822 identified circ RNAs were exonic circ RNAs,and only 345 were intronic circ RNAs.We also compared the expression levels of circ RNA hosting genes with others,the results showed that the averaged expression levels of circ RNA hosting genes were significantly higher than the genes that no detectable circular transcripts in both SSCs and FGSCs.Subsequently,8115 m RNAs,3996 lnc RNAs,and 921 circ RNAs exhibited sex-biased expression that may be associated with germline stem cell acquisition of the sex-specific properties required for differentiation into gametes.Gene Ontology(GO)and KEGG pathway enrichment analyses revealed different functions for these sex-biased lnc RNAs and circ RNAs.We further constructed correlated expression networks including coding–noncoding coexpression and competing endogenous RNAs with bioinformatics.Co-expression analysis showed hundreds of lnc RNAs were correlated with sex differences in mouse germline stem cells,including lnc RNA Gm11851,lnc RNA Gm12840,lnc RNA 4930405O22 Rik,and lnc RNA Atp10 d.Ce RNA network inferred that lnc RNA Meg3 and cir RNA Igf1 r could bind competitively with mi RNA-15a-5p increasing target gene Inha,Acsl3,Kif21 b,and Igfbp2 expressions.These sexbiased expressed lnc RNAs and circ RNAs lay a foundation for future research into the key events(including meiosis,X chromosome inactivation,dosage compensation,etc.)in the process of germ cell development.The female-biased expressed lnc RNA Xist is a biomarker of X chromosome inactivation in mammals.It indicated that one of the two X chromosomes in FGSC was inactive.While oocyte carry two active X chromosomes,so X-chromosome undergoes a process of reactivation in female germ cells from FGSCs to oocytes and we could study the controversial dosage compensation by this process.We used RNA-seq data from mouse germ cells at different developmental stages to compare the expression levels of X-linked and autosomal genes.We concluded that it is not suitable to include genes with no/low expression when measuring X:A ratio(X:A ratio means the ratio of X-linked to autosomal expression as measured by RNA-seq,namely the X:A ratio is indicative of the X:AA ratio when the cell contains one active X chromosome and the XX:AA ratio when the cell contains two active X chromosomes,respectively).We also found that the average gene dosage on the X chromosome always achieved a similar level to autosomes,the median median X:A expression ratio is close to 1 whether there are one or two active X chromosomes present in cells.Next,when single-cell RNA-seq data were analyzed by excluding no-/low-expression genes,these results also supported Ohno’s hypothesis.Ultimately,the analysis of single-cell RNA-seq data across individual cells of mouse preimplantation embryos of mixed backgrounds that we could use strain-specific SNPs to distinguish transcription from the maternal and paternal chromosomes provided direct evidence for dosage compensation.When the paternal X-chromosome was inactive,the average gene dosage on the active maternal X-chromosome increased to achieve a balance between the X-chromosome and autosomes.Taken together,we,for the first time,analyzed and compared the lnc RNA and circ RNA profiles germline stem cells.We found lots of signaling pathways including GDNF singnaling pathway,and plenty of lnc RNA and circ RNA exhibited sex-biased expression that may be association with germline stem cell acquisition of the sex-specific properties required for differentiation into gametes.Meanwhile,our analysis of RNA-seq data(particularly single-cell RNA-seq)from the process of inactivation/reactivation provides direct evidence for dosage compensation.This study provides the theoretical foundation for the researches in germ cell development and human reproduction and so on.