WAC And SC35 Mediate The Existing Mechanism Of R-LOOP

The R loop is a triple-stranded structure composed of a hybrid strand formed by DNA-RNA and a single-stranded DNA in a free state.There are many factors that form the R loop.The lack of DNA-RNA helicase during the transcription process or the RNA formed by the transcription is not processed in time,which will lead to the generation of the R loop.Early research found that the production of R-loops can lead to accumulation of DNA damage and decreased genome stability,and many cancer-related studies have found that many R-loop-related genes are abnormally expressed.With the continuous deepening of the exploration of the R loop,the research found that the R loop is widespread in normal organisms,not only in higher animals,but also in yeast and E.coli.Current research believes that although the R loop has a certain role in promoting the occurrence and development of cancer,some normal physiological activities are also inseparable from the regulation of the R loop.The link between the R loop and some diseases is not as obvious as cancer genes and diseases.The reason why the R loop promotes the occurrence of diseases and cancer is mainly caused by the special structure of the R loop indirectly.The special structure of the R loop can cause DNA double-strand breaks(DSB),gene recombination,and during the transcription process the R loop can also mediate abnormal extension of transcription.The study found that after the formation of the R loop during transcription,in order to maintain the normal progress of transcription and DNA replication,XPF and XPG two endonucleases will excise the R loop from the DNA double strand,which leads to DNA double strand break.At the same time,if the R loop formed during transcription is not properly excised or degraded,it will increase the pressure of subsequent DNA replication and cause the replication fork to stall.The stalled replication fork is a very unstable structure like the R loop,which easily leads to gene recombination and reduces the stability of the genome.In the transcription process,WAC associates RNF20 / 40 with RNAP ?,and promotes H2 B ubiquitination by enhancing the E3 ligase activity of RNF20 / 40 to regulate the transcription process.The study found that in mammals with Bre1(RNF20 / 40)deletion,chromosomal instability(CIN)increased dramatically.CIN is considered to be an early event of cancer,and DSB is the key cause of CIN.At the same time,in recent years,studies have found that many RNA-binding proteins(RBP)affect genome stability during transcription to translation,such as capping enzymes,SRSF1,and SETX.RBP can counteract the negative supercoil formed during the transcription process,thereby inhibiting the production of R loops.In this study,we found that RNF20,WAC,and SC35(SRSF2)inhibit the production of R loops by regulating certain processes of transcription.Through immunofluorescence staining,we found that there is a very obvious interaction between WAC and SC35 in the nucleus of U2 OS cells.At the same time,through immunoprecipitation and immunoblotting,the interaction of endogenous WAC and SC35 was also proved in 293 T cells.In order to explore the specific effects of WAC and SC35 on the R loop,we treated U2 OS cells with siRNA to regulate the expression of specific proteins.At the same time,the R loop was stained with an antibody that specifically recognized the DNA-RNA structure in the R loop to quantify the number of R loops in the nucleus in different experimental groups.SETX,as a DNA-RNA helicase,can separate DNA and RNA during transcription.It is known that after knocking down the expression of SETX protein,the number of R-rings in the cell will increase significantly.Therefore,we used SETX as the corresponding positive control to verify the staining result of S9.6 in U2 OS.The results showed that the number of R-loops in the nucleus of cells treated with siRNA increased significantly,and accompanied by the formation of ?H2AX,which marked an increase in DNA damage.In order to further verify the correctness of S9.6 staining results,we constructed the HA-RNH1(HA-RNASEH1)plasmid.RNH1,as a ribonuclease,can catalyze the hydrolysis of RNA.After the R loop is formed,the overexpressed RNH1 can partially degrade the RNA in the DNA-RNA,making the S9.6 antibody unable to recognize the single-stranded DNA structure.While treating U2 OS with siRNA,we transiently transfected the HA-RNH1 plasmid,and observed the change of the number of R loops in the nucleus by immunofluorescence.The results showed that after overexpressing RNH1,the number of R loops recognized by S9.6 antibody in U2 OS cell nucleus decreased sharply.After confirming the connection between RNF20,WAC,SC35 and R loop,we also verified the connection between R loop and DNA damage.After knocking down the expression of RNF20,WAC and SC35 proteins,we found that the number of ?H2AX in U2 OS cells increased significantly.At the same time,after further treatment with DOX(Doxorubicin,doxorubicin hydrochloride),the number of ?H2AX in the nucleus of U2 OS cells increased more significantly.After discovering that there is an interaction between WAC and SC35,and when the expression of WAC and SC35 is low,there is a significant increase in the number of R loops in the cell.In order to further explore the sites where WAC and SC35 affect the generation of R loops during transcription,we treated 293 T cells with siRNA and performed DRIP-qPCR experiments.The experimental results show that when the expression of WAC and SC35 protein is low,there is a very large overlap of the sites forming the R loop during the transcription process of the two.And in the related ChIPseq experiments,we found that the enrichment regions of WAC and SC35 also have a great overlap.