Study On The Function Of Overlapping Transcription Of SIS Gene Subcluster Of Zebrafish Six2a-OT And Six6b-OT In SIX Gene Cluster

Long non-coding RNA(lncRNA)are defined as transcripts longer than 200 bp but without a coding sequence.Recently,more and more lncRNAs have been identified,and they have been found to regulate gene expression by several different mechanisms.Two lncRNA genes over the SIX gene subcluster were found in The Zebrafish Information Network(ZFIN).Six2a-OT(six2a overlapping transcript lncRNA)gene located across the whole six2a-six3 a clusteron chromosome 13.Six6b-OT(six6b overlapping transcript lncRNA)gene located near six6b-six1b-six4 b cluster on chromosome 20.The length of Six2a-OT gene and Six6b-OT gene is 137837 bp and 154056 bp,respectively.Both of them have four exons.After splicing,the length of Six2a-OT and Six6b-OT mature transcriptional product is 648 bp and 756 bp,respectively.In human,lncRNA HOTAIR has been found playing important role in regulating HOX gene expression.As the positional relation of the two SIX Over Transciptions with SIX gene cluster in zebrafish genome is similar with HOTAIR and HOX gene.We speculate that Six2a-OT and Six6b-OT may be involved in regulating the expression of SIX genes(six2a-six3 a and six6b-six1b-six4b).In qPCR assay,Six2a-OT and Six6b-OT were expressed stronger in zebrafish ovary than in testisThe expression of Six2a-OT and Six6b-OT was higher in the 1-cell stage than in the other periods(6 hpf,24 hpf and 48 hpf).And the expression of Six2a-OT and Six6b-OT decreased gradurally after 6 hpf.Whole mount in situ hybridization was also used to evaluate tissue specific expression of Six2a-OT and Six6b-OT.Six2a-OT gene was abundant in the otic vesicle and spinal cord of the 48 hpf embryo,which suggests that Six2a-OT might be involved in the development of otic vesicle and spinal cord of zebrafish.However,the expression of Six6b-OT was not specific in all the embryonic stages examined.To show the function of Six2a-OT in zebrafish,a specific Six2a-OT antisense morpholino was designed to block the splicing of Six2a-OT.After injecting Six2a-OT morpholino into the cell of 1-cell stage zebrafish embryo,the expression of Six2a-OT decreased significantly comparing with the embryos injecting with standard control morpholino.After knocking down the expression of Six2a-OT,63% of embryos showed short trunk,lateral curvature,or edema at 48 hpf.At the same time,the expression of six2 a and six3 a in Six2a-OT morpholino injection group was significantly lower than that in Standard Control morpholino injection group.Combine with expression pattern,we propose that Six2a-OT may be involved in otic vesicle and spinal development through regulating the expression of six2 a and six3 a in zebrafish.In order to determine how Six2a-OT works with six2a-six3 a gene subclusters,we predict and analyze them by bioinformatics methods and tools.At the same time,the possible interaction between Six6b-OT and six6b-six1b-six4 b is predicted and analyzed.In the part of bioinformatics prediction and analysis in this study: firstly,the secondary structures of Six2a-OT and Six6b-OT are obtained by using RNAfold structure prediction tool.It is found that both of them are folded into complex stem ring structures.This is the basis on which lncRNA can interact with other genes,such as RNA and protein.Then predict the interaction of the two lncRNA with six2 a,six3a and six1 b,six4b,six6 b transcription product mRNAs using the online tool RNAup.The predicted results suggest that the binding free energy of Six2a-OT to Six3 a mRNA is higher.The binding free energy of Six6b-OT and Six1 b mRNA was higher than that of Six4 b or Six6 b mRNA.Finally,lncPro and RPI-Pred were used to predict the possible RPI(RNA-Protein interaction)between Six2a-OT,Six6b-OT and SIX proteins respectively.The predicted results showed that the interaction fraction of Six2a-OT with SIX2 protein was higher than SIX3 protein.The interaction fraction of Six6b-OT with SIX4 protein was higher than that of SIX1 or SIX6 protein.Taken together,Six2a-OT participates in the development of otic vescle and spinal of zebrafish embryos by regulating the expression of six2a-six3 a.However,the molecular mechanism of Six2a-OT and Six6b-OT involved in the regulation of SIX gene subcluster expression will be further explored in future.