Project description:In order to identify genes with different overall transcript levels or differential exon levels (alternative processing) between the groups Control and Tat-SF1KD, we studied 11 hybridizations on the HumanExon10ST array using mixed model analysis of variance. 526 genes with significant transcript level differences between the groups and 1397 genes with significant differential exon levels were found, including 99 genes with both transcript and exon level differences (p<0.01).
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.
Project description:Flp-In T-REx-293 cells transfected with non-targeting control siRNA or UPF1-specific siRNA as indicated and used for total RNA-seq.
Project description:We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB. From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing. Keywords: control / knockdown comparison
Project description:Human umbilical vein endothelial cells (HUVECs) were insulted with cobalt chloride to induce cell apoptosis and alternative splicing in the mimic-hypoxia environment. We use Affymetrix exon array to reveal differential expression from transcript-level and exon-level in genome-wide.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype and UPF3B KO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3A.
Project description:HIV-1 Tat induces the expression of interferon (IFN)-inducible genes in immature dendritic cells (iDC) in the absence of IFN production. We evaluated how three alleles of Tat and some Tat mutants differ in cellular gene modulation and whether a similar gene induction pattern could be detected by treating cells with IFN’s. The three alleles and mutants, with the exception of mutants TatSF21-47 and TatSF2G48-R57A that do not localize in the nucleous, modulated to different degrees IFN-inducible genes without concomitant induction of IFN’s. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all induced genes to a significantly greater extent than full-length Tat. The 2nd exon appears to diminish the gene modulation that can be observed when the first exon alone is expressed. To investigate what domain of Tat are critical to the host-pathogen interactions that are Tat-dependent during HIV infection, we evaluated a variety of Tat-mutants and found that in antigen presenting cells, blood-derived myeloid iDC and macrophages, the second exon of Tat reduces innate immunological responses which are maximal when a Single exon Tat is expressed.
Project description:We knockdowned OMD and PRELP expression by specific siRNAs in 5637 bladder carcinoma cells and conducted expression profile analysis. We also overexpressed OMD and PRELP in T-REx 293 cells and conducted expression profile analysis.
