- Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek ’ s disease virus in vitro. - Long non-coding RNAs (lncRNAs) participate in Marek ’ s disease virus (MDV) replication but how m 6 A modifications in lncRNAs are affected during MDV infection is currently unknown. - We report for the first time profiling of the alterations in transcriptome-wide m 6 A modification in lncRNAs of MDV-infected CEF cells.. - Marek’s disease (MD) induced by Marek’s disease virus (MDV) is a lethal lymphotropic disease of chickens that is characterized by severe immunosuppression, neuronal symptoms and the rapid onset of T-cell lymphoma [1].. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - Expression profiling of long intergenic non-coding RNA (lincRNAs) has also been previously reported in the chicken bursa following MDV infection. - Acting through regulation of the SATB1 gene, the lincRNA linc-satb1 derived from SATB1 was shown to be crucial in the MDV-induced immune response [10]. - All of the RNA modifica- tions are mediated by methyltransferase “writer” com- plex, which is an enzyme complex containing methyltransferase-like 3 (METTL3), METTL4, Wilms’. - As one of the most abundant and conserved RNA modifications, m 6 A is known to be involved in various viral infections, suggesting an important regulatory role in viral replication and pathogenesis [16]. - Alter- ations in the m 6 A signature of lncRNAs suggests that m 6 A modifications may play important regulatory roles during MDV replication.. - Transcriptome-wide m 6 A modifications in lncRNAs after Md5 (a very virulent MDV strain) infection. - RNA-sequencing and transcriptome analyses were performed on mock control and Md5-infected CEF cells following successful construction of cDNA li- braries (Fig. - To gain further information of transcriptome-wide m 6 A modifications in the lncRNAs, we then performed Methylated RNA immu- noprecipitation sequencing (MeRIP-seq). - Using this approach, we identified 363 and 331 m 6 A peaks in the Md5 and control groups, respectively (Fig. - Furthermore, a total of 294 and 275 annotated genes were mapped to the Md5-infected and control groups, respectively (Fig. - Among them, 277 m 6 A peaks and 228 m 6 A modified genes were detected in both the Md5- infected and control groups. - Overall, these results in- dicated that the incidence of the m 6 A modification in lncRNAs was higher in the Md5 infected group com- pared to the control group.. - Results from the methylation heat map and cluster ana- lysis showed that the different clustering could clearly distinguish the m 6 A modification at the transcriptome level in the Md5-infected group from the control group (Fig. - These findings indicate that the degree of methylation in the Md5-infected group was significantly higher than for the control group (Fig. - Regarding the abundance of the m 6 A peaks in lncRNAs, we found that 77.13% of the lncRNAs in the Md5-. - infected group contained m 6 A peaks, which appeared marginally more than the unimodal value calculated at 75.86% in the control group. - peaks being 15.81 vs vs 5.10 and 3.14% vs 2.38%, respectively, for the Md5 infected versus control group (Fig. - To analyze the conserved motif of m 6 A modified lncRNAs, we selected the sequences of the first 1000. - 2 Transcriptome-wide m 6 A modifications in lncRNAs following Md5 infection. - a Venn diagram of m 6 A modification sites identified in lncRNAs from mock control and Md5-infected groups. - b Venn diagram of m 6 A modified lncRNAs from mock control and Md5-infected groups. - The sequence of the top ten peaks with the highest enrichment ratio of lncRNA (50 bp on each side of the vertex) was compared with the motif se- quence found, and it was found that GGACU sequence. - Cluster analysis of the transcriptome (a) and m 6 A modified lncRNA genes (b) in mock control and Md5-infected groups. - The color intensity represents the size of the log-fold enrichment (FE) value. - Notes: Chromsome/ TxStart/ TxEnd: the coordinates of the differentially methylated RNA sites in bed format, please ref http://genome.ucsc.edu/FAQ/FAQformat.html#format1.. - was one of the conserved motif sequences of lncRNA (Fig. - GGACU is one of the motif obtained based on E-value. - In Md5-infected group it was 165/1000.. - The MF outputs showed the genes with increased methylation were notably enriched in the steroid hormone mediated signaling pathway, re- sponse to retinoic acid, nucleosome organization, nu- cleosome assembly, hindbrain development, DNA packaging, chromatin assembly and cellular response to steroid hormone stimulus (Fig. - Notes: Chromsome/ TxStart/ TxEnd: the coordinates of the differentially methylated RNA sites in bed format, please ref. - 4 Differentially methylated N6-methyladenosine peaks in lncRNAs. - Both a and b showed that representative upmethylated genes in Md5- infected group relative to mock control group. - a Number of lncRNA harboring different numbers of m 6 A peaks in the two groups, with the majority harboring only one m 6 A peak. - b The sequence motif of m 6 A sites in Md5-infected and mock control groups. - and c cellular components significantly enriched for the up-methylated transcriptome in Md5-infected versus mock control groups. - important roles in the life cycle of viruses and therefore also in viral pathologies. - Instructively, knockdown of the ALKBH5 demethylase or alternatively the METTL3/14 methylase to alter the level of HIV m 6 A modifications either promotes or in- hibits viral replication, respectively [19]. - However, the impact of the m 6 A modification in MVD is yet to be de- termined [20].. - MDV infection increased lncRNAs m 6 A modification In the present study, we investigated how the m 6 A modification in lncRNAs was affected by MDV infection.. - The results obtained in CEF cells showed that the abun- dance and distribution of m 6 A in Md5-infected and con- trol groups were different albeit not significantly.. - Interestingly, we found that some of the lesser expressed genes in the control group were not only highly expressed in the infected group, but also displayed in- creased levels of m 6 A modification. - GO analysis of the m 6 A modified genes showed that most are up-regulated methylated sites. - LncRNA expression can be variously regulated by his- tone modification, DNA methylation or through changes in the expression of the responsible transcription factors.. - In this study, many differentially expressed m 6 A modifi- cation sites were found, among which the unique m 6 A modification related genes were only found in Md5- infected group. - These results suggest that some of the m 6 A modification sites are changed by Md5 virus infec- tion. - Notably the ErbB gene encoding tyrosine kinases of the epider- mal growth factor (EGF) receptor family can promote. - 7 KEGG analysis and gene set enrichment analysis (GSEA) of differentially methylated genes in Md5-infected and control groups. - Comparing MDV infected and control cells we identified the abundance of m 6 A modifications and the genome wide utilization of the conserved motif. - A very virulent MDV strain, Md5 (Genbank accession no: NC_002229.3) was used in the present study. - After 3′ adaptor-trimming and low-quality reads removing by cutadapt software (v1.9.3), the reads were aligned to the chicken reference genome (Gal5. - To define the possible roles of the differentially meth- ylated genes, the GO functions were analyzed using the corresponding lncRNA genes as inputs. - Genomes (KEGG) [29] analyses of the genes associated with differentially methylated lncRNAs were used as in- puts to derive significantly altered pathways. - MDV: Marek ’ s disease virus. - This work is supported by Grants of the Starting Foundation for Outstanding Young Scientists of Henan Agricultural University (No 30500690). - The grants above were used in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - The datasets generated and/or analyzed during the current study are available in the NCBI repository (https://www.ncbi.nlm.nih.. - Pathogenesis of Marek's disease virus infection. - https://doi.org . - Marek's disease virus: from miasma to model. - doi.org/10.1038/nrmicro1382.. - https://doi.org/10.1016/j.virusres . - https://doi.org/10.1128/. - https://doi.org/10.1128/JVI . - Epigenetic regulation of the latency-associated region of Marek's disease virus in tumor-derived T-cell lines and primary lymphoma. - https://doi.org/10.1128/JVI.06113-11.. - https://doi.org/10.1038/nrg.2 015.10.. - Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript. - Hyperediting by ADAR1 of a new herpesvirus lncRNA during the lytic phase of the oncogenic Marek's disease virus. - https://doi.org/10.1099/jgv.0.. - https://doi.org/10.1038/srep15184.. - Linc-GALMD1 regulates viral gene expression in the chicken. - https://doi.org/10.3389/fgene . - Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. - https://doi.. - org/10.1038/nature11112.. - https://doi.org/10.1038/. - https://doi.org/10.101 6/j.cell . - https://doi.org/10.1038/nsmb.3162.. - https://doi.org/10.1093/bioinformatics/. - Latest insights into Marek's disease virus pathogenesis and tumorigenesis. - https://doi.org/10.3390/cancers12030647.. - https://doi.org/10.1038/s . - https://doi.org/10.1016/j.chom . - https://doi.org/10.1016/j.virol . - T cells in response to Marek's disease virus infection.. - https://doi.org/10.3390/genes10090718.. - https://doi.org/10.1016/j.micinf . - https://doi.org/10.101 6/j.chom . - https://doi.org/10.3390/v12091024.. - org/10.1016/j.vetmic . - https://doi.org/10.1093/nar/gkaa970.
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