Remarkable improvement in detection of readthrough downstream-of-gene transcripts by semi-extractable RNA-sequencing.

The mammalian cell nucleus contains dozens of membrane-less nuclear bodies that play significant roles in various aspects of gene expression. Several nuclear bodies are nucleated by specific architectural noncoding RNAs (arcRNAs) acting as structural scaffolds. We have reported that a minor population of cellular RNAs exhibits an unusual semi-extractable feature upon using the conventional procedure of RNA preparation and that needle shearing or heating of cell lysates remarkably improves extraction of dozens of RNAs. Because semi-extractable RNAs, including known arcRNAs, commonly localize in nuclear bodies, this feature may be a hallmark of arcRNAs. Using the semi-extractability of RNA, we performed genome-wide screening of semi-extractable long noncoding RNAs to identify new candidate arcRNAs for arcRNA under hyperosmotic and heat stress conditions. After screening stress-inducible and semi-extractable RNAs, hundreds of readthrough downstream-of-gene (DoG) transcripts over several hundreds of kilobases, many of which were not detected among RNAs prepared by the conventional extraction procedure, were found to be stress-inducible and semi-extractable. We further characterized some of the abundant DoGs and found that stress-inducible transient extension of the 3'-UTR made DoGs semi-extractable. Furthermore, they were localized in distinct nuclear foci that were sensitive to 1,6-hexanediol. These data suggest that semi-extractable DoGs exhibit arcRNA-like features and our semi-extractable RNA-seq is a powerful tool to extensively monitor DoGs that are induced under specific physiological conditions.

Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles.

Many membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Although these MLOs co-exist in cells, how they maintain their independence without coalescence or engulfment remains largely unknown. Here, we investigated the molecular mechanism by which paraspeckles with core-shell architecture scaffolded by NEAT1_2 long noncoding RNAs exist as distinct MLOs. We identified NEAT1 deletion mutants that assemble paraspeckles that are incorporated into nuclear speckles. Several paraspeckle proteins, including SFPQ, HNRNPF and BRG1, prevent this incorporation and thus contribute to the segregation of paraspeckles from nuclear speckles. Shell localization of these proteins in the paraspeckles, which is determined by NEAT1_2 long noncoding RNA domains, is required for this segregation process. Conversely, U2-related spliceosomal proteins are involved in internalizing the paraspeckles into nuclear speckles. This study shows that the paraspeckle shell composition dictates the independence of MLOs in the nucleus, providing insights into the importance of the shell in defining features and functions of MLOs.