Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours. Total RNA was extracted and global changes in gene expression were determined using microarray chips. MiRNAs are transcribed by RNA pol II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA-box in miRNA and a subset of protein-coding genes is associated with increased sensitivity to a slow rate of transcription elongation. We also show that promoters driven by TATA-box or NF-κB elicit high transcription re-initiation rate, but paradoxically lower levels of miRNA. Interestingly, miRNA synthesis was converted to a more productive mode by decreasing initiation rate, but less productive when the re-initiation rate increased. This phenomenon was found to be associated with a delay in miR-146a induction by NF-κB. We also demonstrate that miRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of miRNA synthesis directed by the TATA-box or NF-κB is a consequence of frequent transcription initiation that lead to Pol II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for miRNA synthesis, and have implications for TATA-box promoters in general. HEK293T cells were transfected with plasmids directing the expression of α-amanitin-resistant variants of Pol II (Rpb1-amr and R749H-amr). α-amanitin was added and RNA was prepared 24 and 48 h later, respectively. The data provided is from 3 Rpb1-amr vs 3 R749H-amr (6 samples).
Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours. Total RNA was extracted and global changes in gene expression were determined using microarray chips. MiRNAs are transcribed by RNA pol II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA-box in miRNA and a subset of protein-coding genes is associated with increased sensitivity to a slow rate of transcription elongation. We also show that promoters driven by TATA-box or NF-κB elicit high transcription re-initiation rate, but paradoxically lower levels of miRNA. Interestingly, miRNA synthesis was converted to a more productive mode by decreasing initiation rate, but less productive when the re-initiation rate increased. This phenomenon was found to be associated with a delay in miR-146a induction by NF-κB. We also demonstrate that miRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of miRNA synthesis directed by the TATA-box or NF-κB is a consequence of frequent transcription initiation that lead to Pol II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for miRNA synthesis, and have implications for TATA-box promoters in general.
Project description:Transcriptional profiling of Murine Embryonic Fibroblasts (MEFs) infected with Ad-MyD88 vs. Ad-GFP or mock infected. Three-condition experiment, Ad-MyD88 vs. Ad-GFP vs. Mock infected cells. Biological replicates: 3 Ad-MyD88, 3 Ad-GFP, 3 mock, independently grown and harvested. One replicate per array.
Project description:Transcriptional profiling of Bone-Marrow derived mouse Dendritic Cells (bmDCs) infected with Ad-MyD88 vs. Ad-GFP or mock infected Three-condition experiment, Ad-MyD88 vs. Ad-GFP vs. Mock infected cells. Biological replicates: 3 Ad-MyD88, 3 Ad-GFP, 3 mock, independently grown and harvested. One replicate per array.
Project description:To identify genes that may regulate distinct or overlapping functions of IRF-3 and IRF-7, primary human macrophage preparations were transduced with adenoviral vectors: Ad-GFP (control), Ad-F3 (expressing the active form of IRF-3, IRF-3 5D), or Ad-F7 (expressing the active form of IRF-7, IRF-7 D247-467) and evaluated by microarray analysis. RNA was collected 24 hours post-transduction with Ad-GFP, Ad-F3 and Ad-F7 and subjected to microarray analysis. Submited tables show the average of 7 donors.