Annotation And Functional Analysis Of Long Noncoding RNA Derived From ERV Key To Early Embryonic Development In Pig

Early embryonic development in mammals is a very complex biological process,which involves the entire stage of fertilization of the oocyte to the development of the blastocyst.With the development of science and medicine,the early embryonic development of mammals has gradually become the focus of attention of many researchers.The research on the early development mechanism of mammalian embryos has a good impetus to the development of assisted reproduction in the medical field.Pigs are similar to humans in immunology and physiology,and the mechanisms of early embryonic development.Therefore,it is urgent to comprehensively study the early embryos of pig.Long noncoding RNAs(lnc RNAs),a class of longer than 200 nucleotides(nt)and non-translated endogenous cellular transcripts,have emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression in cis and/or in trans in the nuclear and/or in the cytoplasmic compartments,and generating an intricate network with RNAs,promoters and enhancers,and chromatin-modifier complexes.The biogenesis of lnc RNAs is associated with endogenous retrovirus(ERV).Long terminal repeats(LTRs)derived lnc RNAs have been evidenced to participate in a wide variety of the biological process,such as evolution,development and disease.Generally,RNA-seq data is mainly used to annotate lnc RNAs.However,due to the limitation of the reads length,the degradation of the samples,the method of library construction and the bias of bases,the annotation is normally incorrect and can not get comlpete sequences,especially loss of the end of transcripts.So,the annotation,the expression quatification and the function analysis will also be incorrect.And,it is important to aquire the accurate information of lnc RNA.It has been reported that small RNAs(s RNA)from promoters in both sense and antisense directions are byproducts of divergently transcribed RNAPII genes,and several other promoter-proximal s RNAs have been discovered.More importantly,a number of s RNA derived from within mature m RNA regions have also been reported in Drosophila and the 3'UTR of He La m RNA.Thus,we persume the combination of RNA-seq and s RNA-seq data will be benefit to annotate the complete transcripts.In this study,we first integrated the RNA-seq and s RNA-seq data(GSE88860)of porcine oocyte and zygote we have obtained preeviously,and annotated the transcripts.27,694 and 32,875 transcripts were found in oocyte and zygote respectively,and s RNAs involved in the annotation of 89.05% of the transcripts and mainly destributed in the 5' and 3' end.To analysis of the completement of the transcripts,we compared the length of the transcripts with and without s RNAs,and found the length of the transcripts with s RNAs were significantly longer,and we also found the initial bases of the the transcripts with s RNAs were A or G.Furthermore,by de novo motif search analysis,we observed TATA box,AATAAA terminater,and the sequence rich in GC at the upstream and downstream of the transcripts with s RNAs,indicating the transcripts annotated with the combination of RNA-seq and small RNA-seq data are mainly complete.Furthermore,6,312 and 18,349 lnc RNAs were found in the transcripts with s RNAs in oocyte and zygote,respectively,and over 20% of them were novel.Consistent with lnc RNAs identified in mouse previously,the length and the expression level of the novel lnc RNAs were shorter and lower than those of coding genes.Then,the lnc RNAs associated with ERVs were screened.30.05% of the lnc RNAs were related with ERV/LTR,and LTRs were mainly destributed in the 5' and 3' end,and they were strongly associated with the ERVK and Ma LR LTR subfamilies,suggesting in mice,lnc RNAs are mainly derived from the LTRs.Also,the length and the expression level of the LTR-lnc RNAs were shorter and lower than those of coding genes.Our results demonstrate the combination of RNA-seq and small RNA-seq data can improve the annotation of transcripts,benefiting to function analysis if LTR-lnc RNAs.In order to analyze the regulation of LTR-lnc RNA on early embryo development in pigs,we focused on LTR-lnc RNA,which is highly expressed in zygotes,and screened a total of 19 LTR-lnc RNAs.LNA-si RNAs targeting on each of them were injected into MII oocytes,and the 19 LTR-lnc RNAs were effectively knocked down.The capacity of the embryonic development was analyzed at 24 h,48h,and 156 h post-IVF.It was shown that after TCONS?00035465 knockdown,the cleavage rate was significantly reduced compared to the control group(p<0.05);after TCONS?00031520 knockdown,the cleavage rate and four-cell rate were not significantly affected,while the blastocyst rate was significantly lower than the control group(p<0.05).Analysis of the expression patterns shown that TCONS?00035465 showed high expression in the two-cell stage,suggesting that it may be related to the zygotic genome activation;TCONS?00031520 showed high expression in the eight-cell stage,indicating that it may have a certain effect on densification and intensification of porcine morula.In summary,in the study,we demonstrated combination of RNA-seq and s RNA-seq data can improve the annotation of complete transcripts,and screened and analyzed the functional LTR-lnc RNAs key to early embryos in pig.The conclusions are as follows:(1)The combination of RNA-seq and s RNA-seq data is benefit to transcript annotation.(2)The expression of LTR-lnc RNAs is higher in zygote than in oocyte.(3)LTR-lnc RNAs TCONS?00035465 and TCONS?00031520 may function on the early embryonic development of pig.