Project description:RNA of total cell, or nuclear fraction, or cytoplasmic fraction was prepared using RNeasy Plus mini Kit. Purified RNA was quantified by Nanodrop (Thermo Fisher). The RNA samples were subjected to transcriptome analysis using Illumina Human BeadChip 22k according to manufacturer’s instructions.
Project description:We performed RNA-protein interaction study iCLIP, i.e single nucleotide resolution uv crosslinking and immunoprecipitation for DNA binding transcription factor CLAMP in different cellular fractions (Chromatin fraction, Nucleoplasmic fraction, Cytoplasmic Fraction) to explore sex-specific RNA binding properties of CLAMP in female (Kc) and Male (S2) cells
Project description:To study isoforms of nuclear RNAs, including CARMN lncRNA, we performed Oxford Nanopore long-read sequencing of RNAs isolated from the nuclear fraction of human coronary artery smooth muscle cells.
Project description:Karyopherin α 2 (KPNA2, importin α1) is involved in the nuclear import of proteins and participates in cellular processes and accumulates in the nucleus under cellular stresses including hydrogen peroxide-indeced oxidative stress via unknown mechanisms. We performed the immunoprecipitation assay and SILAC-based quantitative proteomic analysis in HeLa cells to reveal that hydrogen peroxide decreased the phosphorylation of KPNA2 at serine 62 (S62) and increased its interaction with nucleophosmin 1 (NPM1). Specifically, a reduction in phosphorylated S62 on KPNA2 was observed in the cytoplasmic fraction, but this phosphorylated KPNA2 was not detected in the nuclear fraction. We also identified fifty-four KPNA2-interacting proteins in the nuclear fraction. Among them, NPM1, RPLP0, and MYH9 interacted with KPNA2 in cells exposed to hydrogen peroxide but not in unstressed control cells. Furthermore, the subcellular fractionation, immunoprecipitation, western blotting, immunofluorescence assay, and qRT-PCR analyses were used to support a novel finding that S62 phosphorylation maintains KPNA2 in the cytoplasm, and hydrogen peroxide reduces the phosphorylation of KPNA2 S62 to promote KPNA2-NPM1 complex nuclear entry for gene regulation.
Project description:We isolated RNAs from cytoplasmic or nuclear fraction of dendritic cells, and subject these RNAs for high throughput sequencing. After mapped to mouse genome, sequencing reads (18-30nt) were calculated as the log2 ratio using the normalized TPM (transcripts per million reads) value to determine the expression difference of each sRNA from nuclear and cytoplasmic fractions. We then picked up the superior nucleus-localized sRNAs according to the criteria that abundance of each sRNA was over 20 and x>10y (x: abundance of sRNA from nuclear fraction; y: abundance of sRNAs from cytoplasm fraction).
Project description:The PIWI interacting RNA pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein Gtsf1 is an essential factor for Piwi mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts stably with a sub-population of nuclear Piwi and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9me3 marks at euchromatic transposon insertions and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi interacts productively with a target RNA, resulting in assembly of a silencing complex with Gtsf1 as one core component. impact of loss of DmGtsf1 on transcription and H3K9m3 in ovarian somatic cells (OSC)
Project description:To identify HNRNPA2B1 binding sites on endogenous nuclear RNAs, we performed HITS-CLIP for endogenous HNRNPA2B1 and RNA-seq to analyze the nuclear RNA under either METTL3 or HNRNPA2B1 depletion. Wild type MDA-MB-231 cells were subjected to the HITS-CLIP procedure on immunoprecipitated HNRNPA2B1 associated RNA obtained from the nuclear fraction (Licatalosi D, et al. 2008, Nature 456:464-U22). For RNA-seq, nuclear RNA was extracted from MDA-MB-231 or Hela cells knocked down for METTL3 or HNRNPA2B1.
Project description:Compared to proteins, glycans, and lipids, much less is known about RNAs on the cell surface. We developed a series of technologies to test for any nuclear-encoded RNAs that are stably attached to the cell surface and exposed to the extracellular space, hereafter called membrane-associated extracellular RNAs (maxRNAs). We visualized maxRNAs on the cell surface, sequenced selected maxRNAs, and validated two maxRNAs by RNA fluorescence in situ hybridization. To test for cell-type specificity of maxRNA, we used randomized antisense oligos to hybridize to single-stranded transcripts exposed on the surface of human peripheral blood mononuclear cells (PBMCs). Combining this strategy with imaging flow cytometry, single-cell RNA sequencing, and maxRNA sequencing, we identified monocyte as the major type of maxRNA+ PBMCs and prioritized 11 maxRNAs for functional tests. Extracellular hybridization of antisense oligos to the cell surface-exposed fragments of FNDC3B and CTSS transcripts inhibited monocyte adhesion to vascular endothelial cells. Collectively, these data highlight maxRNAs as a functional component of the cell surface, suggesting an expanded role for RNA in cell-cell interaction and communication.