Our laboratory focuses on developing technologies and exploring the 9DNase digestion, chromatinPreparation & RnaseH treatmentregulatory functions of lncRNAs. Many of lncRNAs reside in the cell nucleus and interact with the genomic DNA in the mammalian genome. In order to analyze the RNA-chromatin interactions, we developed a new tech-nology named RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq) that precisely maps genome-wide RNA-chromatin interactions in intact nuclei. Interactome analysis by RADICL-seq revealed distinct genome occupancy patterns for specific classes of transcripts in each cell type. Interest-ingly, most interactions in cis were from the intronic regions of protein-coding transcripts whereas interactions in trans were from the exonic regions of non-coding transcripts, which indicates the possibility that the intronic regions of protein-coding transcripts play a regulatory role in gene expression. RADICL-seq is expected to be widely used in FANTOM6 and other projects as a valuable technique for understanding the roles of lncRNAs from a spatial perspective.FANTOM6 aims at creating a comprehensive catalogue of functional lncRNAs. As a pilot project, we targeted 285 lncRNAs for a systematic knockdown analysis in human primary fibroblast cells to explore their func-tions. From this analysis we created the largest-to-date publicly available (https://fantom.gsc.riken.jp/zenbu/reports/#FANTOM6) lncRNA knockdown data set on cell growth and morphology using realtime imaging and CAGE deep sequencing to reveal molecular pathways associated with each lncRNA. We found that nearly 30% of the lncRNAs affect cell growth, morphology and migration.A functional lncRNA, named SINEUP, is known to upregulate the produc-tion of a specific protein. We discovered the mechanism by which interaction of SINEUP with a pair of RNA binding proteins, PTBP1 and HNRNPK, allows SINEUP to be transported from the cell nucleus to the cytoplasm, making it possible for it to act on its target mRNA for protein translation. Understanding the mechanisms of functional lncRNAs is an important first step for applica-tions of lncRNAs to improve human health.Figure: Schematic representation of the RADICL-seq protocolTop: Series of enzymatic reactions occurring in fixed nuclei after partial lysis of the nuclear membrane. The adduct formed by genomic DNA (black), RNA (red), and proteins (blue circles) is subjected to controlled DNase I digestion and chromatin prepa-ration. After RNase H digestion, an adapter (dark blue) contain-ing an internally biotinylated residue (black dot) bridges the RNA and DNA molecules that lie in close proximity. Bottom: Series of enzymatic reactions performed in solution. After reversal of crosslinks, the RNA–DNA chimera is converted into a fully double-stranded DNA (dsDNA) molecule and digested by the EcoP15I enzyme to a designated length (adpr, adapter). After ligation of the sequencing linkers (yellow) and biotin pull-down, the library is subjected to PCR amplification and high-throughput sequencing.Recent Major PublicationsToki N, Takahashi H, Sharma H, Valentine MNZ, Rahman FM, Zucchelli S, Gustincich S, Carninci P. SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies. Nucleic Acids Res 48, 11626-11644 (2020)Ramilowski JA, Yip CW, Agrawal S, Chang JC, Ciani Y, Kulakovs-kiy IV, Mendez M, Ooi JLC, Ouyang JF, Parkinson N, Petri A, Roos L, Severin J, Yasuzawa K, Abugessaisa I, Akalin A, Antonov IV, Arner E, Bonetti A, Bono H, Borsari B, Brombacher F, Cameron CJ, Cannistraci CV, Cardenas R, Cardon M, Chang H, Dostie J, Ducoli L, Favorov A, Fort A, Garrido D, Gil N, Gimenez J, … Bail-lie JK, Forrest ARR, Guigó R, Hoffman MM, Hon CC, Kasukawa T, Kauppinen S, Kere J, Lenhard B, Schneider C, Suzuki H, Yagi K, de Hoon MJL, Shin JW, Carninci P. Functional annotation of human long noncoding RNAs via molecular phenotyping. Genome Res 30, 1060-1072 (2020)Bonetti A, Agostini F, Suzuki AM, Hashimoto K, Pascarella G, Gimenez J, Roos L, Nash AJ, Ghilotti M, Cameron CJF, Valentine M, Medvedeva YA, Noguchi S, Agirre E, Kashi K, Samudyata, Luginbühl J, Cazzoli R, Agrawal S, Luscombe NM, Blanchette M, Kasukawa T, Hoon M, Arner E, Lenhard B, Plessy C, Castelo-Branco G, Orlando V, Carninci P. RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chroma-tin interactions. Nat Commun 11, 1018 (2020)Invited presentationsCarninci P. “Fine regulations of cellular networks: novel ap-proaches using antisense lncRNAs” The Human Cell Atlas Developmental Asia and Australia Clinical and rare disease applications seminar (United Kingdom/Online) December 2020Carninci P. “Decoding human genome: an international effort spanning two decades” The 2020 ENCODE Research Applica-tions and Users Meeting (ENCODE 2020) (Spain/Online) October 2020Carninci P. “Analytical technologies for bulk and single cell genomics” The 5th IABS Cell Therapy Conference (Tokyo, Japan) February 2020Carninci P. “Lessons from large consortia: collaborations, inter-nationalization, career paths” EMBO Expert meeting, How do elite programs for early carrer researchers work? (Tokyo, Japan) February 2020Carninci P. “Surprises from studies of mammalian transcrip-tome” The 1st CIBoG Retreat (The 12th NAGOYA Global Retreat) (Nagoya, Japan) February 2020Laboratory for Transcriptome TechnologyTeam Leader: Piero Carninci
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