The Novel Function Of NRF2 In Regulating RNA Splicing And The Mechanism In Cancer

BackgroundRNA splicing is an important biological process of gene expression in eukaryotic cells,but the aberrant isoforms of genes caused by RNA splicing were also associated with many diseases,especially cancer.With the increasing number of abnormal genes caused by altermative splicing in tumor cells,RNA splicing has become to be one of the key factors affecting the tumor,so investigating the function of RNA splicing in tumor provides new insight for cancer prevention and therapy.NRF2(NF-E2 p45-related factor 2)/ARE(antioxidant response element)signaling pathway is an important antioxidant system in the body,a series of genes encoding antioxidant enzymes and phase ? detoxification enzymes can be activated by this pathway for cell defending,thus plays an important role in cancer prevention.But KEAP1 mutations or NRF2 mutations exist in many tumors,leading to a high-level of NRF2 expression in tumors,and inversely protecting the tumor cells by promoting malignant proliferation or improving drug resistance to chemotherapy drugs,indicating theadverse effectof NRF2 in promoting cancer development.Up to now,few studies have addressed the relationship between NRF2 and RNA splicing in cancer.Whether NRF2 expression can affect RNA splicing or influence the splicing of important genes in tumor is still unknown.Objective This research focus on the role of NRF2 in RNA splicing,and its mechanism in cancer.MethodsTo knock out of NRP2 in HeLa cells,CRISPR/Cas9 technique was used.To establishthe stable cell line over-expressing NRF2,lentivirus infection was used.To investigate the function of NRF2 in RNA splicing,high-throughput of RNA sequencing of different cell lines was chose.To detect the impact of NRF2 in SMN expression level,RT-qPCR,luciferase reporter system,western blot were used.Co-immunoprecipitation,GST pulldown,immunofluorescence and tandem affinity purification were used for exploring the interaction between NRF2 and SMN.Gene compensation was used to verify the role of NRF2 in RNA splicing.Results1.NRF2 knockout cell lines and NRF2 overexpressing cell lines were successfully generated in HeLa cells.Aberrant splicing events weregenerated caused by NRF2 knockout,NRP2 knockdown and NRF2 over-expression in tumor cells,including in mRNA splicing and alternative splicing.2.NRF2 regulated the transcriptional level and protein level of SMN:NRF2 can bind SMN promoter and regulate the transcriptional level of SMN.The transcriptional level and protein level of SMN decreased when NRF2 knockdown or knockout;GEMIN2 and COILIN expression were also decreased in NRF2 knockdown or knockout cells.3.NRF2 interacted with SMN in vivo and in vitro:NRF2interacted with SMN in A549 cells.NRF2 and SMN eo-localized in A549 and HeLa cells,the co-localization between NRF2 and SMN existed both under normal condition and stress condition.NRF2 interacted with SNMN complex and localized together in Cajal bodys.Three fragments of SMN(45-90,227-248,268-294)are required for interacting with NRP2,and SMN(268-294)is the essential part for NRF2 binding.The interaction between NRF2 and SMN complex was validated by tandem affinity purification(TAP)assay.4.Gene compensation of NRF2 gene can rescue 1008 aberrant splicing genes and gene compensation of SMN gene can rescue 1106 aberrant splicing genes,and number of overlapping genes is 916,the number of genes only rescued by NRF2 is 92.ConclusionThis study revealed tihat NRF2 regulates RNA splicing,including mRNA splicing and alternative splicing.The molecular mechanism may involve up-regulating the expression levels of SMN complex and through direct interaction with SMN complex in Gems and Cajal bodys.