Identification Of MicroRNAs And The Interaction Pattern With Target Genes In Virus Infected Emiliania Huxleyi

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic phytoplankton which is distributed from sub-polar to tropical latitudes and often forms large mesoscale blooms.E.huxleyi has ornate CaCO₃ extracellular platelets?coccoliths?and the ability of high-yielding of Dimethyl sulfide?DMSP?.In the marine ecosystem,viruses can control the development of blooms and populational dynamic through triggering phytoplankton cell death.The lysis of E.huxleyi affects the carbon dioxide content in the water and atmosphere and aggravates the green house effect due to the release of DMSP into atmosphere and the coccolith formation,abscission,and sedimentation.Moreover,they can facilitate the export of CaCO₃ into seafloor which involved in the carbon cycle of biosphere and geological evolution.The demise of this algal bloom depends on viral infection making it even more important.Therefore,E.huxleyi becomes a key indicator species for the study of biogeochemical cycle and climate change.However,the understanding of the cellular physiological and biochemical reaction,molecular mechanism of viral infection,the biological significance of host-virus interaction is still in its infancy.MicroRNA?mi RNA?is endogenous small RNA which regulates gene expression through an array of sophisticated regulatory networks on transcriptional or post-transcriptional level.It plays important roles in cellular proliferation,differentiation,apoptotic process and immune response to viral infection.The exclusive dependence of viruses on the host cellular machinery for their propagation and survival also make them highly susceptible to the vagaries of the cellular environment.The host can use miRNA encoded by itself to interfere with the proliferation of the virus,and the virus can also fight or modulate the host to meet its needs through a similar mechanism.Therefore,miRNA plays a very important role in the process of virus-host interaction.To date,knowledge concerning the roles of miRNA in phytoplankton is still limited,while the miRNAs of marine phytoplankton in response to virus infection remains to be studied.The recent availability of genomic resources for the E.huxleyi host and its specific virus 86?EhV86?provides an unprecedented opportunity to explore molecular mechanism from the view of miRNA regulation and to gain insights into the biological characteristics of the virus infection.In this study,the miRNAs involved in virus infected E.huxleyi BOF 92 by EhV99B1 were investigated by using Illumina high-throughput sequencing technology combined with molecular cell biology methods.The results are as follows:?1?Forty two miRNAs were identified in small RNA libraries and all of them were novel mi RNAs in which 8 miRNAs were differentially expressed,including 6 host miRNAs?mir1^mir 7?and 2 viral miRNAs?mir 8 and mir 9??mir 5 was eliminated due to its longer length?.The length of these miRNAs was 18 to 30 nt,and predicted 33 precursors ranged from 39⁸5 nt.Approximately 70%miRNAs were produced from exon and intronic regions and exhibited a U bias at the 5'end.These miRNAs were compared to miRBase and E.huxleyi CCMP 1516mi RNAs.No homology indicated that these miRNAs might be unique for E.huxleyi BOF 92 and EhV 99B1.?2?GO and KEGG analysis showed that the target genes of differential expression miRNAs were involved in lipid metabolism,sphingolipid metabolism,autophagy,apoptotic process,photosynthesis,cell cycle and other metabolisms.The interaction networks of differential expression miRNA-mRNA were constructed by using Cytoscape software.The results revealed that the most target genes were regulated by mir 6 and it played an important role in maintenance of the whole network stability owing to its powerful regulatory ability.Moreover,mir 6,mir 7,mir 8 and mir 9,together with other genes,they formed a intricate and intimate network,and these miRNAs inclined to work with more regulatory factors which might play a prominent part in the mentioned metabolisms above.Furthermore,host miRNAs seemed to be more important for cellular regulatory in early viral infection,while the appearance of viral miRNAs mediated regulatory was in the late stage of infection.?3?Quantitative Real-time PCR were used to detect 8 differentially expressed miRNAs and target genes related to autophagy,apoptotic,lipid and sphingolipid metabolism,photosynthesis and cell cycle.The expression trend of mir 6?7?8?9 were up-regulated in EhV-infected samples which were consistent with sequencing data,suggesting these miRNAs were closely related to viral infection.The results of qRT-PCR of these target genes showed that the expression of Cyclin and CKS?related to cell cycle?PBSO?related to photosynthesis?,PSAT and hSPT?related to lipid and sphingolipid metobolism?were significantly down-regulated;while the expression of ATG3,ATG7,ATG13,VPS34?related to autophagy?and metacaspase?related to apoptotic process?were up-regulated.Additionally,partial viral genes including viral endonuclease,DNA polymerase,vSPT and MCP also were up-regulated in comparison with early viral infection.These results revealed the correlation between the expression levels of mi RNAs and their target genes,indicating mi RNAs could be as negative or positive regulators of their targets.?4?The binding-sites of viral miRNAs?mir 8,mir 9?and autophagy-associated genes including ATG3?17282310?,ATG13?17261891?,VPS34?17269912?and VPS34?17275006?were precisely predicted by using miRanda software.The results showed the binding-site polymorphism between these mi RNAs and their targets where miRNAs could bind target genes in 3'UTR,5'UTR or CDS.Subsequently,a serial of the wild type and mutant dual-luciferase recombinant plasmid including psiCHECK2-ATG3-CDS,psiCHECK2-VPS34-CDS,and psiCHECK2-ATG13-3'UTR were constructed based on psiCHECK2 dual-luciferase reporter system.These recombinant plasmids and miRNAs were co-transfected into hepG2 cell.We found that the relative luciferase activity of plasmid containing the 3'UTR of ATG13 was significantly decreased by mir 9,and the relative luciferase activity of plasmid containing the coding region?CDS?of ATG3 and VPS 34 were significantly increased by mir 8 and mir 9respectively.These results suggested that ATG13 probably was the target gene of mir 9,and ATG3,VPS34 probably were the target genes of mir 8 and mir 9 respectively.Furthermore,there was no significant difference for the relative luciferase activity among other transfection groups,indicating the regulatory relationship of them were non-existent.