Identification,Characterization And Function Of A Novel Group Of Natural Antisense Transcripts From RNA1.2 Gene Locus Of Human Cytomegalovirus

Background:Human Cytomegalovirus(HCMV)latent infection,which accounts for more than 50% of the population,is usually manifested as a latent infection in healthy adults,but can cause fatal infection in immunocompromised patients and newborns.HCMV infection of fetus leads to congenital cytomegalovirus(c CMV)infection,which is an important factor endangering the perinatal health of infants.The pathogenic mechanism of c CMV infection has always been one of the research hotspots in the field of virology.cancer is the most common type of endocrine malignancy.HCMV is one of the largest human infectious viruses in the genome.Long and complex genome is predicted to encode 165 open reading frames(ORF).Its complex coding ability includes sense transcription,antisense transcription,variable splicing,non-coding transcription and so on.The non-coding RNA of HCMV(nc RNA)is transcribed from the genome and performs various biological functions at RNA level.At present,the characteristics and functions of nc RNA transcripts of HCMV have not been clearly studied.To clarify the transcriptional structure,transcriptional phase and transcriptional mode of HCMV nc RNA is of great significance to reveal the function and pathogenic mechanism of HCMV nc RNA.Long non-coding RNA(lnc RNA)are RNAs consisting of more than 200 nucleotides and not able to encode proteins.Natural antisense transcription(NAT)is a kind of lnc RNAs.Four lnc RNAs,asr transcribed by HCMV genome,namely RNA1.2,RNA2.7,RNA4.9 and RNA5.0.While their functions and regulatory mechanisms are not completely clear.Besides,there is no report about antisense transcription of RNA1.2 gene region.The purpose of this research is to study the antisense transcription of HCMV genome,focusing on whether there is NATs from the antisense strand of RNA1.2.The existence of the new transcript RNA1.2 ASTs were found and verified by RNA high throughput sequencing(RNA-seq),and its characteristics were analyzed.We want to demonstrate that RNA1.2 ASTs belongs to lnc RNA and explore the possible mechanism of RNA1.2 ASTs participating in RNA transport pathway.Methods: 1.RNA of human embryonic lung fibroblasts(HELF)infected with low passage clinical isolates HCMV HAN strain were collected for RNA-seq,analysis of antisense transcription of HCMV genome and whether there were differences in human gene expression at different time point after infection.2.RNA-seq predicted whether there were new transcripts from the RNA1.2 gene region.3.The gene specific probe of antisense strand of RNA1.2 gene was designed.Northern blotting were performed to identify the transcriptional sequence and transcripts of RNA1.2 ASTs at different stages of virus infection.4.For the transcripts detected by Northern blot,sequence-specific primers were designed to detect the 5' or 3' terminal of RNA1.2 ASTs by RACE(Rapid amplification of c DNA ends).5.Northern blotting was used to test RNA1.2 level after RNA1.2 ASTs downregulation by antisense oligonucleotide(ASO)interfering.6.The biotin-labeled RNA1.2 ASTs probe was designed.RNA pull-down was carried out to detect proteins interacted with RNA1.2 ASTs.7.LC-MS/MS analysis was performed to test RNA1.2 ASTs binding proteins.8.Bioinformatics software enriched and analyzed the function of RNA1.2 ASTs binding proteins,and predicted the function of RNA1.2 ASTs.9.The specific binding protein of RNA1.2 ASTs was verified by Western blot.Results: 1.RNA-seq showed that antisense transcription was common in HCMV genome.There were significant differences in human gene transcription group in different infection stages.2.Sequencing reads,matched with the antisense chain of RNA1.2 gene were detected by RNA-seq,indicating that there may be NATs from RNA1.2 region.3.Three antisense transcripts of RNA1.2 gene were identified by Northern blot in the late RNA of HCMV infection,which were named RNA1.2 AST1,RNA1.2 AST2 and RNA1.2 AST3,with length of 1100 nt,1000nt and 600 nt,respectively.4.3'-RACE showed that 3 'ends of RNA1.2 ASTs were at nt7855 and nt8093,located downstream of a classical poly A signal(nt7848).5'-RACE sequencing showed that 5 'ends of RNA1.2 ASTs were nt7479,nt7331 and nt7203,and there was a typical TATA box(nt7074)upstream.The three newly discovered transcripts were RNA with poly A tail.Bioinformatics software predicted that there were no typical ORFs so that these NATs belonged to lnc RNAs.5.ASO interfered with RNA1.2 ASTs and down regulated its expression,but it did not affect the transcriptional level of RNA1.2.6.The protein mixture with specific binding to RNA1.2 ASTs was obtained by RNA pull-down.7.LC-MS/MS analysis showed that RNA1.2 ASTs could bind 701 proteins.8.GO(Gene ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis showed that RNA1.2 ASTs-binding proteins were involved in biological processes and signal transduction pathways,such as RNA transport,gene silencing,Regulation of viral transcription and so on.9.Western blot verified that RNA1.2 ASTs bind to e IF2-?(Eukaryotic translation initiation factor-2 subunit beta),thus affecting RNA transport.Conclusion: The NATs found from the RNA1.2 gene region of HCMV by RNA deep sequencing belongs to lnc RNA.RNA1.2ASTs do not directly regulate the transcriptional level of RNA1.2 and respond to the transport of RNA by binding to e IF2-?.