Unknown,Transcriptomics,Genomics,Proteomics

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Identification of protein-RNA interactions in Drosophila using RIP-Seq

ABSTRACT: The goal of these experiments is to identify RNAs bound by individual RNA-binding proteins. Drosophila S2R+ cells were transiently transfected with vectors encoding the indicated HA-tagged RNA-binding proteins, or without an insert as a control. Expression was induced with CuSO4, whole cell lysates were prepared and protein-RNA complexes were isolated by immunoprecipitation with HA immunoaffinity resin. RNA was then isolated from the immunoprecipitated material, and RNA-Seq libraries were prepared and sequenced. Sequence reads were aligned to the D. melanogaster reference genome Dm3 using TopHat and expression levels were quantitated using cufflinks. This work was funded as part of the modENCODE project by NHGRI. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf At least two biological replicates were performed for each protein studied. In addition, control experiments were performed in which cells were transfected with the same vector lacking an open reading frame.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Brenton Graveley

PROVIDER: E-GEOD-37756 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

Project description:Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1,YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. Indeed, we show that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central M-bM-^@M-^\switch on/offM-bM-^@M-^] proteins to coordinate the regulation of C. albicans morphogenesis. ChIP was performed in 2 independently grown C. albicans sfl1 or sfl2 homozygous mutant strains expressing (sfl1-CaEXP-SFL1-HA or sfl2-CaEXP-SFL2-HA, respectively) or not (sfl1-CaEXP or sfl2-CaEXP, respectively) SFL1-HA or SFL2-HA (-HA, 3'-triple-HA-tagged alleles of SFL1 or SFL2) under the control of a methionine-repressible promoter (Total samples = 8; 2xCaEXP-SFL1-HA, 2xCaEXP-SFL2-HA, 2xCaEXP control for SFL1-HA ChIP and 2xCaEXP control for SFL2-HA ChIP).

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Identification of protein-RNA interactions in Drosophila using RIP-Seq

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