Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

West, Jason A.; Mito, Mari; Kurosaka, Satoshi; Takumi, Toru; Tanegashima, Chiharu; Chujo, Takeshi; Yanaka, Kaori; Kingston, Robert E.; Hirose, Tetsuro; Bond, Charles S.; Fox, Archa; Nakagawa, Shinichi

doi:10.1083/jcb.201601071

Cited by 289 publications

(385 citation statements)

References 47 publications

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“…Indeed, super-resolution microscopy and crosslinking experiments have confirmed that SGs contain a labile liquid shell. These results point to a complex internal organization of SGs, a picture that, given recent work, is likely true for other membraneless organelles as well [24,25,68]. …”

Section: Materials States Of Membraneless Organelles: Liquids Hydrogementioning

confidence: 57%

Protein Phase Separation: A New Phase in Cell Biology

Boeynaems

Alberti

Fawzi

et al. 2018

Trends in Cell Biology

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Cellular compartments and organelles organize biological matter. Most well-known organelles are separated by a membrane boundary from their surrounding milieu. There are also many so-called membraneless organelles and recent studies suggest that these organelles, which are supramolecular assemblies of proteins and RNA molecules, form via protein phase separation. Recent discoveries have shed light on the molecular properties, formation, regulation, and function of membraneless organelles. A combination of techniques from cell biology, biophysics, physical chemistry, structural biology, and bioinformatics are starting to help establish the molecular principles of an emerging field, thus paving the way for exciting discoveries, including novel therapeutic approaches for the treatment of age-related disorders.

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Section: Materials States Of Membraneless Organelles: Liquids Hydrogementioning

confidence: 57%

Protein Phase Separation: A New Phase in Cell Biology

Boeynaems

Alberti

Fawzi

et al. 2018

Trends in Cell Biology

1,624

1,517

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“…For this purpose, the typical shell and core substructures of paraspeckles were visualized by super‐resolution microscopy in HeLa cells (Fig EV2A and B). Specifically, RNA‐FISH was performed with three antisense RNA probes: two corresponding to the 5′ and 3′ termini of NEAT1_2 , which labeled the shell of the paraspeckle (green signals in Fig EV2A), and one targeting the middle of NEAT1_2 , which labeled the internal area of the paraspeckle (magenta signals in Fig EV2A) (Souquere et al , ; Mito et al , ; West et al , ). All paraspeckles in a cell with an intact shell and core structure were collectively imaged by maximum intensity projection.…”

Section: Resultsmentioning

confidence: 99%

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

et al. 2017

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long 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 improved extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 molecules are present in a single paraspeckle. Another architectural lncRNA,, 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 of correlated 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.

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“…2 and 6, Supplementary Material, Figs S2 and S4). Distinct subdomains are observed in several classes of non-neuronal RNA granules, suggesting that common structural strategies underpin the organization of these diverse cytoplasmic assemblages (39)(40)(41)(42).…”

Section: Fxg Structurementioning

confidence: 99%

Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains

et al. 2017

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Local mRNA translation in growing axons allows for rapid and precise regulation of protein expression in response to extrinsic stimuli. However, the role of local translation in mature CNS axons is unknown. Such a mechanism requires the presence of translational machinery and associated mRNAs in circuit-integrated brain axons. Here we use a combination of genetic, quantitative imaging and super-resolution microscopy approaches to show that mature axons in the mammalian brain contain ribosomes, the translational regulator FMRP and a subset of FMRP mRNA targets. This axonal translational machinery is associated with Fragile X granules (FXGs), which are restricted to axons in a stereotyped subset of brain circuits. FXGs and associated axonal translational machinery are present in hippocampus in humans as old as 57 years. This FXGassociated axonal translational machinery is present in adult rats, even when adult neurogenesis is blocked. In contrast, in mouse this machinery is only observed in juvenile hippocampal axons. This differential developmental expression was specific to the hippocampus, as both mice and rats exhibit FXGs in mature axons in the adult olfactory system. Experiments in Fmr1 null mice show that FMRP regulates axonal protein expression but is not required for axonal transport of ribosomes or its target mRNAs. Axonal translational machinery is thus a feature of adult CNS neurons. Regulation of this machinery by FMRP could support complex behaviours in humans throughout life.

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Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

Cited by 289 publications

References 47 publications

Protein Phase Separation: A New Phase in Cell Biology

Protein Phase Separation: A New Phase in Cell Biology

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains

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