A Study On The Functional Role Of The ERV-associated Enhancer-type LncRNA LncFKBPL In Early Pig Embryonic Development

The early embryonic development of mammals is a highly complex process involving the entirety of the period from oocyte fertilization to blastocyst formation,which encompasses numerous key biological events such as zygotic gene activation and X chromosome inactivation.With the continuous progress of modern medicine,people have become more interested in this process and the underlying functional mechanisms,which will have a significant impact on the advancement of assisted reproductive technologies in the future.Due to ethical and moral issues,early human embryos cannot be used directly for research,thus alternative species embryos are sought after.Research has shown that pigs are physiologically and embryonically similar to humans,making them a suitable experimental model for studying embryonic development.With the advancement of sequencing technology,a large number of long non-coding RNAs(lncRNAs)have been discovered in early embryos,and they can play a certain regulatory role.LncRNAs are transcripts longer than 200 nucleotides that do not encode proteins,but they share some characteristics with mRNAs,such as being predominantly transcribed by RNA polymerase Ⅱ.LncRNAs play a role in processes such as cell cycle regulation,epigenetic regulation,and dosage compensation effects.In mice and humans,more than two-thirds of lincRNAs are related to endogenous retroviruses(ERVs),which are retrotransposons that belong to transposable elements.ERVs are named for their similarity to exogenous retroviruses,as they contain long terminal repeat(LTR)sequences at both ends.Once integrated into the genome,LTRs can act as promoters or enhancers to activate the expression of neighboring genes,or as terminators to terminate gene transcription,resulting in lncRNA production.Many ERV-related lncRNAs have been found in early embryo development,but research on their specific functional mechanisms is still scarce.It is of certain research value to find ERV-related lncRNAs that may participate in the regulation of early embryonic development.We designed semi-random primers targeting pig ERV sequences and used semi-random amplification to enrich unknown transcripts containing ERV sequences from pig embryos at different developmental stages.We selected a transcript located upstream of FKBPL,which was specifically highly expressed at the 4-cell stage in pigs,through expression pattern analysis of these unknown transcripts.We named this transcript lncFKBPL.Next,we obtained the full-length sequence of lncFKBPL through RACE experiments and found that it had two variable transcription start sites.After comparing with the UCSC website,we found that lncFKBPL contained ERVL,SINE,and LINE sequences.Furthermore,through open reading frame prediction,we found that lncFKBPL did not have a clear open reading frame,so we tentatively judged that lncFKBPL was a poly(A)-tailed lncRNA related to transposon sequences such as ERVL,SINE,and LINE.Subcellular localization analysis showed that lncFKBPL was located in the cell nucleus and mainly located on chromatin,indicating that lncFKBPL was a lncRNA.In order to investigate the function of lncFKBPL,we used siRNA interference to knock down lncFKBPL,and found that the development of pig early embryos was arrested at the 8-cell stage after lncFKBPL interference,indicating that lncFKBPL is essential for pig early embryo development.In order to explore the mechanism by which lncFKBPL regulates early pig embryo development,and because many lncRNAs function through cis-regulatory effects,we first analyzed the effect of interfering with lncFKBPL on the expression of surrounding genes.The results showed that interfering with lncFKBPL led to a decrease in the expression level of the downstream gene FKBPL,while the expression levels of other surrounding genes did not change significantly.Additionally,we found that overexpression of lncFKBPL did not affect the expression levels of FKBPL and other surrounding genes,indicating that lncFKBPL may specifically regulate the expression of FKBPL through a cis-regulatory mechanism.To verify that lncFKBPL is regulated in a cis-regulatory manner,we used the CRISPR/dCas9-mediated endogenous activation system to activate lncFKBPL,and found that endogenous activation of lncFKBPL promoted the expression of the FKBPL gene without affecting other surrounding genes,indicating that lncFKBPL specifically regulates the expression of FKBPL in a cis-regulatory manner.Based on this,we proposed the hypothesis that lncFKBPL regulates FKBPL expression through an enhancer model.To verify this hypothesis,we used a dual luciferase reporter system,and found that whether the lncFKBPL gene sequence was linked upstream or downstream of the reporter system,and whether it was linked in the forward or reverse direction,it could enhance the expression of luciferase,indicating that lncFKBPL can indeed act as an enhancer to regulate the expression of target genes.Furthermore,we found that interfering with lncFKBPL disrupted its enhancer activity,indicating that the enhancer activity of lncFKBPL is mediated by the transcribed lncRNA.In order to further verify the enhancer model,we used gel shift and immunoprecipitation to detect whether lncFKBPL can interact with the MED8 component of the Mediator complex that mediates enhancer activity.The experimental results showed that lncFKBPL can form an RNA-protein complex with MED8.These results suggest that lncFKBPL regulates the expression of FKBPL through an enhancer model.We found that interfering with FKBPL also blocked pig early embryo development at the 8-cell stage,similar to the effect of interfering with lncFKBPL.This suggests that lncFKBPL regulates pig early embryo development by upregulating FKBPL expression.The results of this study reveal some of the functions of lncRNAs in pig early embryo development and provide new references for the mechanism of pig early embryo development research.