Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes

ABSTRACT: Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis using whole exon DNA microarrays integrating alternative splicing and differential gene expression. Purified human CD2+ T or CD19+ B cells were activated using protocols to model the early events in post-transplant allograft immunity and sampled as a function of time during the process of immune activation. Here we show that 3 distinct classes of alternatively spliced and/or differentially expressed genes change in an ordered manner as a function of immune activation. We mapped our results to function-based canonical pathways and demonstrated that some are populated by only one class of genes, like integrin signaling, while other pathways, such as purine metabolism and T cell receptor signaling, are populated by all three classes of genes. Our studies augment the current view of T and B cell activation in immunity that has been based exclusively upon differential gene expression by providing evidence for a large number of molecular networks populated as a function of time and activation by alternatively spliced genes, many of which are constitutively expressed. Keywords: T cells, B cells, alternative splicing, exon CD2+ T-lymphocytes and CD19+ B-lymphocytes were purified from peripheral blood mononuclear cells (PBMC) of 10 normal human donors. Freshly isolated, resting CD2+ T-cells were resuspended in RPMI-1640 complete media and activated with CD3/CD28 Dynal beads. Cultures were sampled at 24, 48 and 72hrs. A subset of isolated, resting cells immediately stabilized by RNALater solution were used as the baseline comparison (Time 0). Freshly isolated CD19+ magnetic-bead purified resting B cells were resuspended in RPMI-1640 complete media to which anti-CD40 antibody was added, followed by cross-linking with goat-anti-mouse anti-IgG1. The cells were then cultured with rIL2 and rIL10 (100 ng/ml each) to simulate the T-cell dependent B cell activation of an alloimmune response. RNA was extracted at time 0, 24, 48 and 72 hours post activation using the mirVana miRNA Isolation Kit. The GeneChipÂ® The RNA was converted into labeled cDNA using the GeneChipÂ® WT Sense Target Labeling kit (Affymetrix). Labeled cDNA was hybridized to Affymetrix Human Exon 1.0 ST arrays. Data for mRNA transcript profiles were generated in the form of CEL files using standard protocols (http://www.affymetrix.com/).

ORGANISM(S): Homo sapiens

SUBMITTER: Sunil Mathan Kurian

PROVIDER: E-GEOD-14352 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Genome-wide analysis of immune activation in human T and B cells reveals distinct classes of alternatively spliced genes.

Grigoryev Yevgeniy A YA Kurian Sunil M SM Nakorchevskiy Aleksey A AA Burke John P JP Campbell Daniel D Head Steve R SR Deng Jun J Kantor Aaron B AB Yates John R JR Salomon Daniel R DR

PloS one 20091119 11

Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion/exclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis of activated human T and B lymphocytes using whole exon DNA microarrays integrating alternativ ...[more]

PMID: 19936255

Similar Datasets

Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells

Dataset Information

Genome Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes

Publications

Genome-wide analysis of immune activation in human T and B cells reveals distinct classes of alternatively spliced genes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure