Study Of The Functions And Mechanisms Of FUS-associated Long Non-coding RNAs

Long noncoding RNAs(lncRNAs)are transcripts with the length of more than 200 nucleotides,which do not have a traditional open reading frame and play a regulatory role at the RNA level.With the development of sequencing technology and intensive research,more and more lncRNAs have been discovered and proved to play important roles in a variety of physiological and pathological processes.Due to the wide variety and complexity of lncRNAs,there are still a lot of problems need to be explored in lncRNAs field.LncRNAs play roles with its binding proteins.Fused in sarcoma(FUS)is an RNA-binding protein that involved in a variety of biological processes including gene transcription,alternative splicing and phase separation by binding to different types of RNA.Previous studies have found that lncRNA binds to FUS protein and participates in gene transcriptional regulation.When cervical cancer HeLa cells sense DNA damages,lncRNA ncRNACCND1s,derived from the cell cycle protein induced CCND1 promoter regions,were induced,and then recruit FUS to CCND1 promoter region.Further,FUS interacts with histone acetylation enzyme CBP/P300,and inhibits the enzyme activity of CBP/P300,resulting in inhibition of CCND1 expression.However,the mechanism of other lncRNAs involved in the regulatory process of FUS remains unclear.In this study,I focused on two FUS-associated lncRNA-LINC00473 and Lhx1os.LINC00473 is a lncRNA molecule that is highly expressed in breast cancer cells.Accumulating evidence has shown that LINC00473,as an oncogene,promoted the development of multiple human cancers.However,the role of LINC00473 in breast cancer has not been reported.Our study found that LINC00473 promoted proliferation of breast cancer MCF-7 cells.Studies have shown that LINC00473 regulates cAMP/CREB target genes in non-small cell lung cancer,but the molecular mechanism remains unclear.Therefore,we identified several cAMP/CREB target genes and found that LINC00473 promoted the expression of cAMP/CREB target gene CCND1.Then,ChIP experiment showed that LINC00473 promotes CCND1 expression through enhances the recruitment of pCREB and histone acetylation(H3K27ac)level at CCND1 promoter.Based on the study in HeLa cells,we speculated that LINC00473 may regulate the expression of CCND1 through ncRNACCND1s/FUS in breast cancer MCF-7 cells.The results showed that LINC00473 can positively regulate ncRNACCND1s in MCF-7 cells.We also found that LINC00473 can bind FUS proteins.These results suggest that LINC00473 inhibits the expression of CCND1 through the ncRNACCND1s/FUS signaling pathway.Therefore,our study revealed that LINC00473 regulates the balance of the pCREB/H3K27ac pathway and the ncRNACCND1s-FUS pathway,results in promoting the transcription of CCND1 properly in breast cancer cells.This work provides a significant reference for the role of lncRNA and FUS in gene transcriptional regulation.In addition,another Fus-associated lncRNA Lhx1os is highly expressed in mouse cerebellum.Interestingly,we found that Lhxlos can form G-quadruplex structure.Then,the results suggested that the G-quadruplex could interact with Fus,while the non-G-quadruplex mutant reduced the interaction with Fus.In recent years,Fus phase separation is hot fields.In vitro,we have verified that Fus proteins formed phase separation.However,how G-quadruplex RNA affects Fus phase separation is not clear.In this study,we performed sedimentation analysis and droplets formation experiment with different molar ratios of G-quadruplex RNA and Fus.The results found that Fus phase separation is promoted with low-concentration of G-quadruplex,while the promotion effect of FUS phase separation is gradually eliminated with the further increase of G-quadruplex concentration.Compared with G-quadruplex,the effect of non-G-quadruplex mutant on Fus phase separation was significantly reduced.When the G-quadruplex structure was disrupted by TMPyP4 in granule cell,the Fus proteins were found to accumulate as foci in the cytoplasm.Normally,Fus is mainly localized in the nucleus.Studies have shown that high concentrations of RNA in the nucleus inhibit Fus phase separation.Many researchers have proposed that Fus neurological aggregation in the cytoplasm is linked to phase separation of Fus.We speculated that the disruption of G-quadruplex destroys G-quadruplex-mediated Fus-RNA interaction,leading to Fus phase separation and aggregation in the cytoplasm.Therefore,our results demonstrated that the G-quadruplex structure formed by a cerebellum highly expressed lncRNA Lhx1os regulated Fus phase separation,and our findings may provide new insights into the regulatory mechanisms of Fus phase separation.In addition,we found a mutation of Lhxlos in mouse neuroblastoma N2a cells during the molecular cloning experiment,named Lhx1os-m.Lhx1os-m promotes the proliferation of N2a cells.Compared with Lhx1os,the binding between Lhx1os-m and Fus was decreased.We speculated that Lhxlos-m might change the RNA structure,such as G-quadruplex,resulting in decreased binding with Fus,which suggested the importance of Lhx1os structure for Fus binding.In conclusion,the studies of Lhx1os and LINC00473 have added new theoretical knowledge to the lncRNA field,and expanded the understanding of the molecular mechanism between lncRNA and FUS protein.