Abstract
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 subnuclear 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.