Project description:To test the effect of silencing Rae1 on expression on RNA polymerase II transcripts, host mRNAs were analysed by cDNA microarrays. We hypothesized that if silencing Rae1 expression increases cellular resistance to inhibition of transcription in VSV infected cells, mRNA characteristic of host antiviral response would be increased than compared to cells transfected with control siRNA. HeLa cells transfected with siRNA targeted against Rae1 (siRae1) or against non-targeting siRNA (siNT). The sequence of nontargeting (NT) siRNA is scrambled and does not match any sequence on the human genome. Transfected cells were either infected with VSV or mock infected. Six hours later RNA was extracted from the cells. Two separate experiments of such were analyzed by microarrays
Project description:Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in many processes in RNA metabolism. In addition to their functions in the nucleus, hnRNPs can function in the replication of RNA viruses in the cytoplasm. In vesicular stomatitis virus (VSV)-infected cells, several hnRNPs relocalize from the nucleus to the cytoplasm. This raises the question of whether these hnRNPs are relocalized together with their host nuclear RNAs or whether they associate with new RNAs in the cytoplasm. hnRNP A1, hnRNP C1/C2, and hnRNP K were immunoprecipitated from mock- or VSV-infected cells, and RNAs were analyzed by high content RNA sequencing. Each hnRNP displayed a loss of interaction with cellular transcripts in favor of viral mRNAs. hnRNP A1 was preferentially associated with VSV phosphoprotein (P) mRNA; hnRNP C1/C2 was preferentially associated with VSV glycoprotein (G) mRNA, and hnRNP K bound viral transcripts in rank of abundance.
Project description:Benzo(a)pyrene (Bap), a key component of cigarette smoke (CS), is a pollutant and widely present in our daily life. The roles of Bap on viral replication have been amply discussed and controversial. In our present study, we investigated the effect and mechanism of Bap on the replication of vesicular stomatitis virus (VSV) and respiratory syncytial virus (RSV) in vitro. HeLa cells were pre-exposed to Bap at 10 μM for 48 h and then infected with VSV (MOI=1) for 24 h. RNA sequencing analysis was conducted to identify Bap-dysregulated genes and qPCR analysis was used to identify the result. Chronic exposed to Bap significantly inhibited the replication of VSV and RSV in vitro and in vivo.
Project description:By performing m6A-seq analysis on the peritoneal macrophages that derived from ALKBH5-/- mice and littermate mice infected with or without vesicular stomatitis virus (VSV), we want to investigate whether ALKBH5 deficiency-mediated m6A RNA methylation contributes to the regulation of its target genes expression. m6A-seq analysis revealed enriched and specific m6A peaks on the transcript of ALKBH5-targeted gene, which were substantially increased in ALKBH5-deficient peritoneal macrophages than that in wild-type cells whatever infected with or without VSV. Meanwhile we didn’t observe up-regulation of m6A signal on VSV in ALKBH5-deficient cells; and also didn't find significant difference of m6A signals on IFN-β mRNA between ALKBH5-deficient and wild type cells whatever infected with or without VSV. These demonstrated that deficiency of ALKBH5 controls viral replication by increasing the m6A modification of ALKBH5 target gene to regulate its expression.
Project description:To investigate the function of XAF1 in chromatin openning. Wild-type or XAF1 knockout HT29 cells were infected with VSV for 0 or 3 hours, ChIP-seq was did with H3K27Ac antibody. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27Ac in HT29 cells.
Project description:To have a global picture of the miRNAs regulated upon treatement with secretome of Salmonella infected cells, we assessed small RNA changes, by RNA-sequencing, of HeLa cells treated with sectretome of Salmonella infected cells or mock-treated cells
Project description:5-methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. We obtained ~1,000 candidate m5C sites in the wider transcriptome, most of which were found in exonic regions of mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCap and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in mRNA and noncoding RNA. Candidate m5C sites in mRNAs are enriched in 5’UTRs and near start codons, and commonly embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that non-conversion levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Altogether, these findings emphasise the major role of NSUN2 in making this mark transcriptome-wide and further substantiate a functional interdependence of cytosine methylation level with mRNA translation
Project description:We have evaluated the response of M14 cells to VSV virus expressing eGFP or insect virus B2 protein. We find interferon related genes to be at higher levels when cells are infected with VSV-B2
