title: Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs.
abstract: NEAT1_2long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improvedNEAT1_2extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional methodNEAT1_2was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50NEAT1_2molecules are present in a single paraspeckle. Another architectural lncRNA,IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in subnuclear granule-like structures. The semi-extractability ofNEAT1_2correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.
authors.affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Chujo
authors.affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Tetsuya
authors.identifier: 0000-0003-1394-5129
authors.initials: T
authors.lastname: Kawaguchi
authors.affiliation: Brain Science Institute, RIKEN, Wako Saitama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kurosaka
authors.affiliation: Brain Science Institute, RIKEN, Wako Saitama, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takumi
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nakagawa
authors.affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo Hokkaido, Japan hirose@igm.hokudai.ac.jp.
authors.forename: Tetsuro
authors.identifier: 0000-0003-1068-5464
authors.initials: T
authors.lastname: Hirose
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D009698:Nucleoproteins / Q000032:analysis / Q000302:isolation & purification; D011485:Protein Binding; D062085:RNA, Long Noncoding / Q000032:analysis* / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA
chemical_list: D009698:Nucleoproteins; D062085:RNA, Long Noncoding
keywords: RNA extraction; RNA‐binding protein; long noncoding RNA; nuclear body; prion‐like domain