Project description:The METTL3-METTL14 heterodimer is the core component of the N6-methyltransferase complex (MTC) that catalyzes methylation of adenosine residues at the N(6) position of RNA. As the non-catalytic subunit of MTC, METTL14 functions as the RNA-binding scaffold that recognizes the RNA substrate. To identify METTL14 binding sites in the transcriptome, we overexpressed HA-tagged METTL14 in HepG2 cells and performed PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) using HA antibody. Subsequent deep sequencing identified 5,961 METTL14 binding sites, in which the GGAC motif was enriched. Overall design: PAR-CLIP using HA antibody in HepG2 cells with HA-METTL14 overexpression
Project description:RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs. Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were sequenced. mRNA half-life data under GSE56751.
Project description:This study was designed to look for the RNAs that directly bind with QKI Overall design: HA tagged QKI-5 and QKI-6 were overexpressed in QPP GSCs and were applied to PAR-CLIP protocol. RNAs were exacted and sent for sequencing with Illumina HiSeq2000 Sequencer.
Project description:This experiment was designed to indentify RNAs making direct contact with EZH2 in mouse embryonic stem cells E14 with an integrated transgene encoding HA-EZH2 were pulsed with 4-SU, irradiated with UV, and subjected to HA immunoprecipitation.
Project description:Following implantation, mouse epiblast cells transit from a naïve to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells (mESC) as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for the exit from naïve pluripotency and the progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naïve pluripotency expression program through the decommissioning of active enhancers associated with key naïve pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow the reactivation of relevant genes required for proper PGC specification. Our findings uncover a wave of activation-deactivation of Foxd3 as a crucial step for the exit from naïve pluripotency and subsequent PGC specification. Genome-wide binding profiles for Foxd3 were investigated in mouse embryonic stem cells (mESC). A mESC line (FH-Foxd3 mESC line) expressing exogenous Foxd3 tagged with Flag and HA epitope (FH-Foxd3) at nearly endogenous levels was generated. ChIPs were performed against FH-Foxd3 using anti-HA or anti-Flag antibodies.
Project description:BACKGROUND:Next-generation sequencing technologies have profoundly impacted biology over recent years. Experimental protocols, such as photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP), which identifies protein-RNA interactions on a genome-wide scale, commonly employ deep sequencing. With PAR-CLIP, the incorporation of photoactivatable nucleosides into nascent transcripts leads to high rates of specific nucleotide conversions during reverse transcription. So far, the specific properties of PAR-CLIP-derived sequencing reads have not been assessed in depth. METHODS:We here compared PAR-CLIP sequencing reads to regular transcriptome sequencing reads (RNA-Seq) to identify distinctive properties that are relevant for reference-based read alignment of PAR-CLIP datasets. We developed a set of freely available tools for PAR-CLIP data analysis, called the PAR-CLIP analyzer suite (PARA-suite). The PARA-suite includes error model inference, PAR-CLIP read simulation based on PAR-CLIP specific properties, a full read alignment pipeline with a modified Burrows-Wheeler Aligner algorithm and CLIP read clustering for binding site detection. RESULTS:We show that differences in the error profiles of PAR-CLIP reads relative to regular transcriptome sequencing reads (RNA-Seq) make a distinct processing advantageous. We examine the alignment accuracy of commonly applied read aligners on 10 simulated PAR-CLIP datasets using different parameter settings and identified the most accurate setup among those read aligners. We demonstrate the performance of the PARA-suite in conjunction with different binding site detection algorithms on several real PAR-CLIP and HITS-CLIP datasets. Our processing pipeline allowed the improvement of both alignment and binding site detection accuracy. AVAILABILITY:The PARA-suite toolkit and the PARA-suite aligner are available at https://github.com/akloetgen/PARA-suite and https://github.com/akloetgen/PARA-suite_aligner, respectively, under the GNU GPLv3 license.
Project description:Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.
Project description:RNA-binding proteins (RBPs) regulate numerous aspects of gene expression; thus, identification of their endogenous targets is important for understanding their cellular functions. Here we identified transcriptome-wide targets of Rbfox3 in neuronally differentiated P19 cells and mouse brain by using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP). Although Rbfox3 is known to regulate pre-mRNA splicing through binding the UGCAUG motif, PAR-CLIP analysis revealed diverse Rbfox3 targets including primary microRNAs (pri-miRNAs) that lack the UGCAUG motif. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs. In vitro analyses revealed that Rbfox3 functions as a positive and a negative regulator at the stage of pri-miRNA processing to precursor miRNA (pre-miRNA). Rbfox3 binds directly to pri-miRNAs and regulates the recruitment of the microprocessor complex to pri-miRNAs. Our study proposes a new function for Rbfox3 in miRNA biogenesis.