Researchers in Japan say they have developed a novel bioinformatic pipeline to define semi-extractable RNAs across human cell lines. Their findings “Landscape of semi-extractable RNAs across five human cell lines” appear in Nucleic Acids Research.
The study provides new perspectives on exploring the involvement of RNA in biological processes such as cancer development and progression, viral RNA degradation, and cellular stress responses, and may drive the development of therapeutic strategies for cancer and infectious diseases, according to the scientists.
“Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method and can be efficiently retrieved by needle shearing or heating during RNA extraction. Semi-extractable RNAs are promising resources for understanding RNA-centric phase separation. However, limited assessments have been performed to systematically identify and characterize semi-extractable RNAs,” write the investigators.
“In this study, 1074 semi-extractable RNAs, including ASAP1, DANT2, EXT1, FTX, IGF1R, LIMS1, NEAT1, PHF21A, PVT1, SCMH1, STRG.3024.1, TBL1X, TCF7L2, TVP23C-CDRT4, UBE2E2, ZCCHC7, ZFAND3 and ZSWIM6, which exhibited consistent semi-extractability were identified across five human cell lines. By integrating publicly available datasets, we found that semi-extractable RNAs tend to be distributed in the nuclear compartments but are dissociated from the chromatin. Long and repeat-containing semi-extractable RNAs act as hubs to provide global RNA–RNA interactions.
“Semi-extractable RNAs were divided into four groups based on their k-mer content. The NEAT1 group preferred to interact with paraspeckle proteins, such as FUS and NONO, implying that RNAs in this group are potential candidates of architectural RNAs that constitute nuclear bodies.” 
“Using the newly developed bioinformatic analysis pipeline, we examined original experimental data from cultured human cell types and successfully identified and characterized 1,074 semi-extractable RNAs potentially involved in the formation of phase-separated membraneless organelles,” explained Chao Zeng, PhD, junior researcher and an assistant professor in the faculty of science and engineering at Waseda University.
Upon investigating the localization of semi-extractable RNAs in chromatin as well as within the cell, the team found that these RNAs were enriched in repressed and repetitive heterochromatin (darkly staining) regions, especially in Polycomb-repressed areas. Inside the cells, the RNAs were concentrated in the nucleus, including the nucleolus, but disassociated from the chromatin.
Additionally, the researchers postulated that the semi-extractable RNAs could potentially function as a platform for interacting with other RNAs. To verify their hypothesis, they compared semi-extractable RNAs with close to 600 hub RNAs forming protein-mediated RNA-RNA interactions with multiple other RNAs. They found that semi-extractable RNAs indeed acted as hubs and were pivotal in forming RNA-RNA interactions.
Further analysis of semi-extractable RNA revealed a marked preference of RNA-binding proteins in binding to AU-rich regions associated with the RNAs. While messenger RNAs typically exhibit the AU-rich regions at the 3’ end, which regulates RNA stability, semi-extractable RNAs exhibited a concentration of AU regions at the 5’ end, indicating potential involvement in undiscovered functions.
The study provides the first dataset of semi-extractable RNAs across human cell lines, which is a valuable resource for investigating RNA-based phase separations.
“Future integration of semi-extractable RNAs with RNA interaction studies will provide insights into the molecular mechanisms underlying RNA-induced phase separation in cells,” said Michiaki Hamada, PhD, professor in the faculty of science and engineering at Waseda University.
Takeshi Chujo, PhD, assistant professor from Kumamoto University, and Tetsuro Hirose, PhD, professor from Osaka University were also involved in the study.
