Identification And Functional Analysis Of ERV-derived LncRNAs During Cellular Reprogramming In Mice

Long noncoding RNAs(lncRNAs),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 lncRNAs is associated with endogenous retrovirus(ERV).ERVs derived lncRNAs have been evidenced to participate in a wide variety of the bilogical process,such as evolution,development and disease.Among sequencing-based techniques to annotate RNA transcripts,next-generation RNA sequencing(RNA-seq)is commonly used.However,because it is unable to handle the ends of a given gene,especially the low expressed ones,identifying only their most extreme boundaries at best,so it constrains the functional annotation of ERV-associated lncRNAs(ERV-lncRNAs).A number of small RNAs derived from the 5' and 3' UTR of mRNAs have been reported in Drosophila,HeLa cells,T cells and embryonic stem cells.These studies raise the exciting possibility that RNA-seq combined with small RNA-seq could allow for the determination of 5' and 3' ends of low expressed transcripts.Exogenous expression of pluripotent transcription factors leads to cell fate conversion,and produce pluripotent stem cells.In 2006,Yamanaka et al.reprogrammed mouse embryonic fibroblasts(mefs)to induced pluripotent stem cells(iPSCs)by Oct4,Sox2,Klf4 and c-Myc(OSKM).The activity of ERV during iPSC reprogramming is dynamic changes,and IncRNAs have been evidenced to work in the process.However,the role of ERV-derived lncRNAs(ERV-lncRNAs)on iPSC reprogramming still need to be understood.In the study,we took advantage of publicly available RNA-seq and small RNA-seq data(GSE102518)from iPSC reprogramming system,which results in near 100%efficiency of iPSCs reprogramming within 7 days by OSKM induction,to perform transcript annotation by RNA-seq and small RNA-seq combined strategy(RSCS).The results are below:1)63,772 transcripts were annotated by RSCS,and sRNAs were involved in the annotation of 92.4%of the transcripts and mainly destributed in the 5' and 3' end.2)We compared the length of the transcripts with and without sRNAs,and found the length of the transcripts with sRNAs were significantly longer,and we also found the initial bases of the the transcripts with sRNAs were A or G.Furthermore,by de novo motif search analysis,we observed TATA box,polyadenylation signal AATAAA,and the sequence rich in GC at the upstream and downstream of the transcripts with sRNAs,indicating the transcripts annotated by RSCS are mainly full-length.3)The boundaries and length distribution of transcripts annotated by RSCS show a genuine representation similar to RefSeq,indicating the complete and precise transcriptome can be generated by RSCS.4)13,072 lncRNAs were found in the transcripts with sRNAs,and 2,711 of them were novel.Consistent with lncRNAs identified in mouse previously,the length and the expression level of the novel lncRNAs were shorter and lower than those coding transcripts.40.8%of the lncRNAs were related with TE.Among them,59.3%were ERV-lncRNAs,and they were strongly associated with the ERVK and MaLR LTR subfamilies.The length and the expression level of the ERV-lncRNAs have no significant difference with ERV-unassociated lncRNAs.5)In agreement with the previous reports,we observed the activity of ERV-lncRNAs was high during the process of iPSC reprogramming,and exhibited specific expression pattern.By analysis of ATAC-seq and ChIP-seq on pluripotency factors and histone modifications,we demonstrate the enhancer associated ERV-lncRNA,TCONS-00000162,could regulate the expression of Prdm14 during iPSC reprgramming.6)To gain insight into the function of ERV-lncRNAs on the cellular reprogramming,we established the Oct4/Nanog-specific module,and investigated the ERV-lncRNAs associated with the regulatory network.The ERV-lncRNAs were highly activated in reprogrammed mefs(rmefs)at day 7 after the induction,and decreased in iPSCs,suggesting their potential function on the process of reprogramming.7)We found the knockdown of lnc703 resulted in the significant decreasion of AP positive colonies,demonstrating its function on iPSC reprogramming.Subcellular localization analysis showed lnc703 was specifically localized in the cytoplasm,revealing it may work as ceRNA.8)Definitly,we found lnc703 poccesses the binding sites of miRNAs targeting Nanog,and its knockdown led to the downregulation of Nanog expression.Furthermore,the deficiency of reprogramming led by knockdown of lnc703 could be rescued by Nanog overexpression.The results demonstrate that lnc703 may sustain the expression of Nanog by sponging the miRNAs to improve the reprogramming efficiency.In summary,in the study,we demonstrated the RSCS can improve the annotation of full-length of transcripts,and screened and analyzed the functional ERV-lncRNAs important to cellular reprogramming in mice.Our results point out the regulatory role of enhancer associated ERV-lncRNA on gene expression.In addition,we show a high prevalence of miRNA-mediated interactions between their mRNA targets and lncRNAs,proposing that this mechanism of ceRNAs indeed functions on regulation of gene expression,particularly in the context of cell-fate transitions.The findings of this study are help to understand the lncRNA,and reveal the function of ERV during mammalian cellular reprogramming.