Project description:The protein Fused in Sarcoma undergoes liquid-liquid phase separation. To investigate whether this phase transition alters the RNA interactome we purified phase-separated FUS droplets and soluble FUS from HEK 293T cells transfected with GFP-tagged WT FUS or P525L FUS. Phase separated FUS was purified by fluorescence-activated particle sorting (droplets) and soluble FUS by co-Immunoprecipitation (IP) respectively followed by isolation of co-purified RNA. Here we show that phase separation affects the RNA interactome of FUS.
Project description:Proximity labeling approaches have been widely utilized to define protein interactomes. Due to the inherent promiscuity of proximity labeling using TurboID-based approaches, identification and adoption of appropriate labeling controls is a pivotal step to mitigate background interference and enhance interactome assignment accuracy. Here, we evaluate the effectiveness of both expression controls and data normalization strategies in generating high confidence interactome maps. This dataset contains proximity labeling proteomics results, including the proximity labeling section of TurboID-HUWE1 in the 293T cell line. It serves as the raw data for Figure 5 in the article.
Project description:To characterize the function of MED23 on transcription, we generated two MED23-KD 293T cell lines in which MED23 was about 75% knockouted by CRISPR-Cas9. Then we performed RNA-seq to analyze the change of transcript levels around termination sites.
Project description:To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection
Project description:To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection 293T cells were seeded on 10cm plates and were grown in DMEM containing 10% FBS under standard TC conditions. Next day, the cells were transfected either by pCMV-Aire (6ug/plate) or empty pCMV (6ug/plate) using Mirus reagent, according to manufacturer's instructions. Total RNA was extracted 48 hrs post transfection by Trizol protocol according to the manufacturer's instructions. 3 biological replicates were used for Aire transfection and 2 replicates for transfection with a control plasmid.
Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples with different treatment (different digestion size and/or crosslinking method) and one (1) MEF sample were included to test our new approach for RNA-interactome mapping and the different samples were analyzed to show RNA interactome differences between them.
Project description:Transcriptional profiling of Human Embryonic Kidney(293T) Cells comparing control untreated 293T cells with 293T cells transfected with A) pcDNA 3.0(-) vector[Invitrogen] (Mock) and B) Expression vector pcDNA 3.0(-) containing cloned Influenza virus H5N1and H11N1-NS1 (Non-Structural1) gene.