Functions Of Circular Rna Circ-S7?-?654-1292 And Circ-S₅?-?1088-1360 Derived From Grass Carp Reovirus

Circ RNA?circular RNA?is a hot research topic in recent years.It is a covalently closed single-stranded RNA molecule.Compared with ordinary linear RNA,circ RNA is more resistant to exonucleases or RNase R,more stable.Circ RNA can play a role by binding with mi RNA and protein,regulating gene transcription and encoding protein.With the development and progress of high-throughput sequencing technology,more and more circ RNAs have been found in archaea,fungi,plants,animals,and humans.Recently,it has also been discovered that?herpes virus and hepatitis B virus can also encode circ RNA.Grass carp reovirus?GCRV?is the pathogen of grass carp hemorrhagic disease.Previous research on GCRV mainly focused on virus-encoded protein genes and virus-host interactions,but there were no reports of circ RNAs derived from GCRV.1.Research on GCRV-encoded circ RNAs based on high-throughput sequencing technologyIn order to explore the potential of GCRV to form circ RNAs,CIK cells infected with GCRV-873 strain were collected for circ RNA sequencing.The sequencing data were compared with GCRV genome sequences to obtain 32 GCRV-derived circ RNAs,of which 31 were derived from the antisense strand of GCRV genomic ds RNA segments.Conservation analysis of 5 nucleotides upstream and downstream of the splice sites of circ RNAs using Web Logo3 software showed that the 2 nucleotide sequences upstream and downstream of the 5'-splice sites were mostly"AG?GG".The two nucleotide sequences upstream and downstream of the 3'-splice sites were mostly"AG?GT".Circ RNA-mi RNA-m RNA network analysis results showed that GCRV circ RNA can interact with host and viral mi RNA to regulate the expression of host genes.These regulated host genes were involved in biological regulation and cell communication,response to stimulus,ion binding,nucleic acid binding and other GO items.In the host m RNA that GCRV circ RNA regulated by interacting with the host mi RNA,no regulated genes were found to be enriched in the KEGG pathway;in the host m RNA that GCRV circ RNA regulated by interacting with viral mi RNA,the regulated genes were significantly enriched in activation of MAPK,the adipocytokine signaling pathway.2.Functions of GCRV circ RNA circ-S₇?-?654-1292In order to explore the function of GCRV circ RNA,two circ RNAs were selected for the study.The sequence of circ RNA circ-S₇?-?654-1292 corresponds to the GCRV S7 segment-/nt:654-1292 region?Gen Bank accession No.AF403393.1?of GCRV-873strain,the PCR were respectively performed with divergent primers and primer targeted junction site,Sanger sequencing of PCR products confirmed that circ-S₇?-?654-1292 is indeed a circular molecule;immunofluorescence results showed that circ RNA circ-S₇?-?654-1292 was mainly distributed in the cytoplasm;q RT-PCR results showed that the abundance of circ RNA circ S₇?-?654-1292 increased with the progress of GCRV infection;biological information analysis results showed that 8 host genes related to immune,apoptosis,and transcriptional regulation may regulated by interaction of circ-S₇?-?654-1292 with 8 GCRV-encoded mi RNAs;transient transfection studies showed that circ-S₇?-?654-1292 significantly up-regulated the expression levels of CD8?and IFN-?genes and inhibited the expression of GCRV VP7 gene,suggesting that circ-S₇?-?654-1292 can control the proliferation of GCRV;According to the results of RNA-Protein Pull-Down,circ-S₇?-?654-1292 can interact with NF-?B,and found that circ-S₇?-?654-1292 can increase the expression levels of p65 and c-Rel genes and promote the transfer of NF-?B to the nucleus,inhibit the progression of CIK cells from G1 to S phase under GCRV infection,and partially reverse the apoptosis induced by GCRV;biological information analysis found that circ-S₇?-?654-1292 has an open reading frame that encodes a small peptide with 29 amino acid residues.Western blotting confirmed that Ds Red protein can be detected when the open reading frame is replaced with a Ds Red sequence.It is speculated that circ-S₇?-?654-1292 has the ability to encode a small peptide consisting of 29 amino acid residues.3.Functions of GCRV circ RNA circ-S₅?-?1088-1360The sequence of circ RNA circ-S₅?-?1088-1360 corresponds to the-/nt:1088-1360region?Genbank accession No.AF403391.1?of the genomic ds RNA S5 segment of GCRV-873 strain,the PCR were respectively performed with divergent primers and primer targeted junction site,Sanger sequencing of PCR products confirmed that circ-S₅?-?1088-1360 was indeed a circular molecule.The results showed that circ-S₅?-?1088-1360 is mainly distributed in the cytoplasm,and its expression level increased with the progress of viral infection.It was found that circ-S₅?-?1088-1360 can up-regulate the expression levels of CD8?,CD4L and IFN-?,activate the immune system of grass carp and suppress the expression of GCRV VP7 gene.It is speculated that circ-S₅?-?1088-1360 can regulate GCRV proliferation.In summary,GCRV RNA can form circ RNAs.circ-S₇?-?654-1292 can up-regulate the expression levels of CD8?and IFN-?genes,interact with NF-?B and promote the transfer of NF-?B to the nucleus.circ-S₇?-?654-1292 can control the proliferation of GCRV,regulate cell cycle,inhibit cell apoptosis,and have the potential to encode a small peptide with 29 amino acid residues.circ-S₅?-?1088-1360 can also up-regulate the expression levels of CD8?,CD4L and IFN-?,and regulate the proliferation of GCRV.These results deepen the understanding of the genetic information of GCRV genome and provide new clues for understanding the relationship between GCRV and its host.