Journal
EMBO JOURNAL
Volume 36, Issue 10, Pages 1447-1462Publisher
WILEY
DOI: 10.15252/embj.201695848
Keywords
long noncoding RNA; nuclear body; prion-like domain; RNA extraction; RNA-binding protein
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [26113002, 26291001, 15K18474, 16H06316, 16K13110]
- Institute for Genetic Medicine, Hokkaido University
- Japan Society for the Promotion of Science
- Grants-in-Aid for Scientific Research [15J01473, 16K13110, 15K18474, 26113005, 16H06316, 16H06463, 17H03604, 26291001, 26113002, 26113001] Funding Source: KAKEN
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NEAT1_2 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 NEAT1_2 extraction by 20-fold (a property we term semi-extractability), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules 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 sub-nuclear granule-like structures. The semi-extractability of NEAT1_2 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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