| In recent years,endogenous small interfering RNAs(endo-siRNAs)a class of small RNA have been found in mammalian germ cells.Endo-siRNA usually 20-22 nt in length,combined with protein Ago2,participate in RNA interference(RNAi)mechanism.Endo-siRNAs are processed from long double stranded RNA(dsRNA)precursors,digestion by DICER.Current research shows that endo-siRNAs are derived from transposable elements,complementary annealed transcripts,and hairpin RNAs.Endogenous retrovirus(ERV)is a major transposon,account 8% and 10% respectively in human and mouse genomes.ERV also plays an inportant role in the early development of human and mouse embryos.The flank of ERV has a long terminal repeat structure(LTR),it can also functioned as promoter and terminator.LTR transcript product can forming dsRNAs,afterwards,generate endo-siRNAs.In our previous study,we used small RNAs high throμghput depth sequencing technology,identified a lot of endo-siRNAs in zygote of pigs.In this study,we focused endo-siRNAs origined from LTRs(LTR-siRNAs),and their effects on early embryonic development of pig.We first verified high-throμghput sequencing results with stem loop real-time PCR.The results show that the LTR-siRNAs are expressed in porcine zygote.We injected Locked Acid(LNA)of Dicer into MII stage oocytes,to knwodown Dicer effectively,and also,the expression of LTR-siRNAs were significantly decreased,indicating the formation of endo-siRNAs is DICER dependent.Endo-siRNAs derived from dsRNA.Single stranded RNA can be degraded by RNaseA,which does not affect dsRNA.We performed RNA-FISH test using LTR fluorescent probes.The result showed a strong fluorescence signal in the embryos of RNaseA treatment group.It demonstrates that the transcriptional products of LTR can form dsRNA.Further we injected LNA-iLTRs to knowdown the expression level of LTRs in zygote,and we observed the expression level of LTR-siRNAs significant reduced.These results showed LTR-siRNAs derived from dsRNA produced by LTR transcripts.To elucidate the regulatory role of LTR-siRNAs on the early development of porcine embryos,firstly,we examined the expression patterns of LTRs and LTR-siRNAs by real-time PCR.The results showed that LTR-siRNAs might inhibit the expression of LTRs in early embryonic development.Moreover,we injected mimics and inhibitors of LTR-siRNAs to the stage of MII oocyte to verify the results.Finally,we injected mimics and inhibitors of LTR-siRNAs and LNA-iLTRs to the stage of MII oocyte,to detected the effects of LTR-siRNAs and their target LTRs on early embryonic development.The results showed that the cleavage rate and blastocyst rate decreased significantly in the mimics and inhibitors of LTR-siRNAs and LNA-iLTRs-injected groups compared with the control groups.The results demonstrate that the LTR-siRNAs play an important role on early development of embryos in pig,by regulating the activation of their targeted LTRs. |
