Analysis to evaluate the presence of 5’ cap in intragenic lncRNAs
ABSTRACT: RNA aliquots were digested with 5’-phosphate-dependent exonuclease (5'-exo) following pre-treatment with tobacco acid pyrophosphatase (TAP) to release the 5’ cap and render the RNA susceptible to 5’ exonuclease digestion (TAP+/5'-exo+). Two color array hybridizations were performed and TAP+/5'-exo+ samples were compared with TAP-/5'-exo+ control samples. Two-condition experiment, TAP+/5'-exo+ vs. TAP-/5'-exo+ treated total RNA samples. Biological replicates: 4 with dye swap
Project description:To identify the sequences responsible for recruitment of Glucocorticoid receptor (GR) to individual loci, we performed ChIP-seq and ChIP-exo that combines chromatin immunoprecipitation with an exonuclease digestion step. We performed these experiments in three cell lines : IMR90 (ATTC:CCL-186), U2OS osteosarcoma cell lines, K562 (ATCC:CCL243), upon glucocorticoid treatment.
Project description:This study involves the role of yeast mRNA decay factors in transcription. The experiment included here are the ChIP-exo results of three decay factors: Xrn1, Dcp2 & Lsm1. Four experiments were made: Xrn1, Dcp2, Lsm1 and control (no-TAP tag), in two replicates.
Project description:TAP-GluN1 (840 kDa and 1.5 MDa), PSD95-TAP (1.5 MDa) and WT (control; 1.5 MDa) native complexes from Grin1^TAP/TAP, Dlg4^TAP/TAP, and WT mouse forebrain (cortex and hippocampus), respectively. Purified samples were seperated by blue native PAGE. Bands were excised, digested with trypsin. Peptides were identified by LC-MS/MS.
Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5’-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-cap reactions were performed with TAP, and without TAP as a control.
Project description:We determined DNA-binding sites of the yeast transcription factor Yfl052w by ChIP-exo. Cells were grown in the YP media containing palatinose. Yfl052w was tagged with HA tag and anti-HA antibody was used for the immunoprecipitation. Examination of Yfl052 trancription factor in HA-tagged and wt cells (as a control)
Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).
Project description:Heat shock protein 90 (HSP90) is a highly abundant molecular chaperone that interacts with many other intracellular proteins to regulate various cellular processes. However, compositions of the HSP90-interacting complex remain underinvestigated. This study thus aimed to characterize such complex in human embryonic kidney (HEK293T) cells under normal physiologic state using tandem affinity purification (TAP) followed by protein identification using an ultrahigh-resolution tandem mass spectrometer (Qq-TOF MS/MS). A total of 32 proteins, including four forms of HSP90 and 16 novel HSP90-interacting partners, were successfully identified from this complex using TAP control to subtract non-specific binders. Co-immunoprecipitation followed by immunoblotting and immunofluorescence co-staining confirmed the association of HSP90 with known (HSP70, α-tubulin, and β-actin) and novel (vimentin, calpain-1, and importin-β1) partners. Knockdown of HSP90 by small-interfering RNA (siHSP90) caused significant changes in levels of HSP70, α-tubulin, β-actin, vimentin, and calpain-1, all of which are calcium oxalate (CaOx) crystal-binding proteins that play significant roles in kidney stone formation. Moreover, crystal-binding capability was significantly decreased in siHSP90-transfected cells as compared to non-transfected control and siControl-transfected cells. In summary, we report herein a number of novel HSP90-interacting proteins in renal cells and demonstrate the potential role of HSP90-interacting complex in kidney stone formation.
Project description:We report ChIP-seq and ChIP-exo data for GR in liver tissue isolated from WT and GRdim mice. Comparison of the mouse models reveals that GR interacts with the genome as both a monomer and dimer. Examination of GR, RNAPII and CEBPb binding in WT and GRdim mice on a genome-wide scale
Project description:Erythroid development and differentiation from multiprogenitor cells to red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, co-operate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are positionally organized with respect to each other and their cognate DNA binding site across the mouse genome remains unclear. We applied high resolution ChIP-exo to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak-pairing, were used to determine high confidence binding locations by ChIP-exo. We identified ~10,000 GATA1 and ~15,000 TAL1 locations, which were essentially confirmed by ChIP-seq. Of these, ~4,000 locations were bound by both GATA1 and TAL1. About three-quarters of these were tightly linked (<40 bp away) to a partial E-box located 7-8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs, that reflect on their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7-8 bp upstream of a WGATAA motif across thousands of genomic locations. Genome wide analysis of GATA1 and TAL1 in G1E and G1E-ER4 cells using ChIP-exo experiments
Project description:The human mitochondrial degradosome is a complex of the ribonuclease PNPase (Q8TCS8, encoded by PNPT1 gene) and RNA helicase SUV3 (Q8IYB8, encoded by SUPV3L1 gene). The aim of the project was to identify proteins which co-purify with both subunits of the degradosome. To this end we used human 293 cells stably expressing hSuv3 or PNPase with a C-terminal TAP tag or TAP tag fused to a mitochondria targeting sequence (control bait) (cell lines are described in details in (PMID: 19864255). Mitochondria were isolated from the cells, lysed and the protein extracts were subjected to affinity chromatography. Co-purified proteins were identified by mass spectrometry and their amounts were quantified by a label-free approach (MaxQuant). These experiments are part of the project “Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria”.