Project description:We employ multi-step affinity purification followed by high-throughput sequencing to determine the location of EJC complexes assembled on a cellular transcriptome in Drosophila S2 cells, finding 6% of the intron-containing genes were not associated with EJCs, and within genes with multiple introns, only specific exon-exon junctions assembled an EJC. RIP-Seq, 3 samples
Project description:We employ multi-step affinity purification followed by high-throughput sequencing to determine the location of EJC complexes assembled on a cellular transcriptome in Drosophila S2 cells, finding 6% of the intron-containing genes were not associated with EJCs, and within genes with multiple introns, only specific exon-exon junctions assembled an EJC.
Project description:We performed lariat sequencing to profile the diversity of spliced RNA lariats in S. pombe identify annotated and alternate introns. Three different growth conditions were used to grow S. pombe wt and S. pombe Δdbr1. Lariat sequencing of the Δdbr1 strains and RNAseq of the wt and Δdbr1 strains were done to profile intron lariats and exon-exon junctions in RNA transcripts.
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.
Project description:Nucleosome organization influences gene activity by controlling DNA accessibility to transcription machinery. Here, we develop a chemical biology approach to determine mammalian nucleosome positions genome-wide. Using this strategy, we uncover surprising new features of nucleosome organization in mouse embryonic stem cells. In contrast to the prevailing model, we observe that for nearly all mouse genes a class of fragile nucleosomes occupies previously designated nucleosome-depleted regions around transcription start sites and transcription termination sites. We show that a subset of DNA-binding proteins including insulator CTCF and pluripotency factors co-occupy DNA targets with nucleosomes. Furthermore, we provide in vivo evidence that promoter-proximal nucleosomes, with the +1 nucleosome in particular, contribute to the pausing of RNA Polymerase II. Lastly, we find a characteristic preference for nucleosomes at exon-intron junctions. Altogether, we establish an accurate method for defining the nucleosome landscape, and provide a valuable resource for studying nucleosome-mediated gene regulation in mammalian cells.
