Citation
- Authors: Yoneda R. et al.
- Year: 2021
- Journal: Int J Mol Sci 22 11014
- Applications: in vitro / lncRNA / jetMESSENGER
- Cell type: HAP1
Method
HAP1 cells were transfected with 30 pmol RNA fragments with JetMESSENGER (150-001, Polyplus, Illkirch, France) according to the manufacturer’s protocol. After 4 h of incubation, the media was changed to fresh new media, and the cells were subsequently used for ICC or cell viability assay.
RNA fragments were transfected to HAP1 cells in order to validate the effect of m6A-modified RNAs on LLPS of TLS/FUS in the cell. Cy5-labeled Fragment 6 was transfected to HAP1 cells:
- Cy5 signals indicated that RNA fragments were transfected to most of the cells, which reflects the high transfection efficiency of the experiment.
- Transfected RNAs formed particles mainly in the cytoplasm and some in the nucleus, but WT TLS/FUS still localized to the nucleus, and did not colocalize with RNAs in the cytoplasm
Abstract
Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS-RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.