Transcriptomic Analysis,Annotation And Functional Assessment Of LncRNA In Porcine Early Embryos

Early embryonic development in mammals refers to the process from the zygote to the blastocyst,which shows a widely similarities among different species,but species specificity still exists in some aspects,such as lineage differentiation,establishment of pluripotency and spatiotemporal regulation of transcription factors.At present,the research on early embryonic development of mammals is mainly limited to model animals represented by mice,and seldom in large mammals,which seriously hinders the understanding of early embryonic development in mammals.Long noncoding RNA(lnc RNA)is a genomic transcription product more than 200 nt and without ability of protein-coding,and can be divided into intragenic lncRNAs and intergenic lncRNAs(linc RNA),accoding to its derivation.Several research suggests that lnc RNA can regulate gene expression at multiple levels,and participate in many biological events such as transcriptional regulation,epigenetic modification and X chromosome dose compensation.In recent years,there are many reports on the functional mechanism of lnc RNA,but almost in mice.In consideration of the poor conservation of lnc RNA among species,it is essential to demonstrate the functional role of lnc RNA in other mammals.Pig,as an important livestock,is considered as human disease model and potential organ donor for xenotransplantation in regenerative medicine,and shows conserved principles of early development with human.In this study,we established a transcriptomic profile of MII oocytes and early embryos,from zygote to early blastocyst in pig,and analyzed the key biological events in the early embryonic development based on high-throughput single cell RNAsequencing technology(scRNA-seq).Furthermore,we annotated a large number of lncRNAs,and the regulation mechanism of linc-321 on early embryonic development of pig was analyzed.The results are as follows:(1)Using the scRNA-seq data from the porcine early embryos,we identified 19,813 genes and established a transcriptomic profile of porcine early embryos,and found that the transcriptome features of pig early embryos were highly stage-specific.The precursor cells of inner cell mass(ICM)and trophoblast(TE)were identified by hierarchical clustering in morula cells.Meanwhile,we found CARM1,YBX3 and RRAS could be ICM marker,while TEAD4,DAB2,NUDT4 and CLDN8 could be TE marker in pig.Further,we analyzed the expression patterrns of pluripotency factors from 8-cell to ICM,which suggest that morula and ICM shows significant na?ve pluripotency characteristics in pig,and found the pluripotency in pig may be dependent on the activity of the JAK-STAT and PI3K-AKT signaling pathways.And,ESRRB,TFCP2L1,UTF1 and OTX2 can be used as na?ve pluripotency marker in pig.We found that the LIF/STAT3,MAPK and PI3K-AKT signaling pathways were highly active in ICM,and the high activity of LIF/STAT3 signaling pathway in 2-cell and ICM was verified by immunofluorescence.The decreasing of regulators associated with repression modifications such as DNA methylation and H3K9me3,and the increasing of regulators associated with activation modifications such as H3K4me3 occur at the switch of 4-cell to 8-cell stages,demonstrating the regulatory role of epigenetic remodeling on ZGA in pig.Then,we identified the sex of embryos by the gene expression level of Y chromosome,and the gene expression level of X chromosome in female and male embryos were analyzed respectively,and the lack of dosage compensation in morula and the initiation of the dosage compensation in ICM were observed.We believe that the dosage compensation may be independent on XIST-induced X chromosome inactivation during early embryonic development in pig.The study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine PSCs and improve research in regenerative medicine.(2)Using the scRNA-seq data from the porcine early embryos,we identified a total of 41,235 novel lncRNAs,and most of them(68%)were linc RNAs.By knocking down the linc RNAs highly expressed in the 1-cell stage specificially,we found that the knockdown of linc-321 would lead to the development arrest at 1-cell stage in pig.Furthermore,we found that the male and female pronuclear envelope fusion(PEF)was abnormal in the embryos after the knockdown of linc-321.By the prediction of linc-321 regulated genes and the verification of Q-PCR,we shown that linc-321 can inhibit the expression of MYT1.RNA-FISH showed that linc-321 was mainly located in the nucleus,suggesting that linc-321 may regulate the expression of MYT1 at transcriptional level.Ch IP-PCR showed that the knockdown of linc-321 resulted in the significant deletion of H3K27me3 modification at the promoter region of MYT1.Based on the prediction of linc-321 binding proteins by RPISeq,we found that linc-321 might specifically interact with SUZ12(PIP-SVM = 0.93),an important component of PRC2.Furthermore,RIP-PCR verified the interaction between SUZ12 and linc-321.The above results showed that linc-321 can recruit PRC2 and establish H3K27me3 at MYT1 promoter to inhibite the expression of MYT1,and promote the pronuclear envelope fusion to form zygote.As the first functional linc RNA characterized in pig,linc-321 provides the clues for investigating the strictly regulated process of early embryonic development.In summary,we described the key biological events during porcine early embryonic development,and the first lineage specification and the emergence of pluripotency are deonstrstated using the scRNA-seq data.The comprehensive analysis provides a new understanding of the first cell fate decision,and is crucial for derivation of na.ve ESCs from porcine embryo.In addition,we comprehensively annotated lnc RNA in porcine early embryos and explored the function and regulatory mechanism of specific linc RNA.The study is a basis towards comprehending the role of linc RNA on large mamalian early embryo.